Clinical Whole Genome Sequencing: Not Quite Ready for Prime Time?

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When I posted “Why I Don’t Want to Know My Genome Sequence here in November 2012, I got a lot of grief. Still do.

Now researchers at Stanford University have put whole genome sequencing (WGS) of genetically healthy folks to a limited but telling test, and the results appear in this week’s Journal of the American Medical Association. (My version’s at Medscape.)

I can’t improve on the clear and compelling language of the JAMA article:

“In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable WGS findings.”

I’m not surprised. DNA science, any science, is by nature uncertain.

WGS can identify certain well-studied “actionable” gene variants, such as those that raise risk of cancers and clotting disorders, and drug sensitivity genes, although all that information may be overkill. Both exome (just the protein-encoding part, 1-3% or so of the genome) and WGS have solved medical mysteries in selected populations: babies in neonatal intensive care units, kids with developmental delay, and people with  undiagnosed diseases.

THE STANFORD DOZEN
At Stanford, the dozen presumably healthy adults met with a genetic counselor and gave blood, from which the white cells yielded DNA. The paper doesn’t go into how the participants were recruited, whether they had consecutive clinic appointments, were hovering near a vending machine, or were part of a zumba class.

Then two teams of experts scrutinized the DNA data.

First, the genome whisperers (three genetic counselors, three physician-informaticists, and one molecular pathologist) used software to look for clues to health-related genes (things like variant frequencies, function predicted from structure, and evolutionary conservation). They scoured the medical literature and a staggering number of gene variants listed in databases, such as those of the Human Gene Mutation Database (HGMD) and the American College of Medical Genetics and Genomics (ACMG).

This meticulous matching of gene variant (aka allele or mutation) to function, the figuring out or finding of what genes do, is called annotation. It’s the difficult, time-consuming part that doesn’t make it into genohype. A genome is sequenced – and then what? Genetic counselors and other genetics experts are paid to hunt down what the variants do and could mean for a person’s health.

Once the genome whisperers applied their criteria to identify DNA sequences that might be meaningful, a team of physicians turned their suggestions into medical advice – tests and referrals, on average three per participant. And just as the 12 people were average Joes and Jills, three of the docs were in primary care, two of whom hadn’t dealt with genetics or genomics before, and the other two were medical geneticists, although all were academics. The study also compared two sequencing platforms – Illumina for all, Complete Genomics for some. (Disclosure: I’ve accepted chocolate at meetings from both companies.)

chromosomesWHAT THE GENOME WHISPERERS FOUND
Here are some interesting findings:

• The sequencing missed 10 to 19% of known inherited disease genes. This is due to incomplete coverage. To derive a genome sequence, many copies are overlapped. The more copies, the more of the genome is represented in the derived sequence. Sequencing in this study missed some genome parts. It happens. But if it happens in the clinic, it can mean a false negative.
• Both platforms approached 100% accuracy in detecting genotypes already known to cause disease. It’s easier to find a unicorn if you know what one looks like.
• The platforms were much better at detecting the well-known single nucleotide polymorphisms (SNPs) than copy number variants – tiny deletions and duplications. That’s because a repeat of a sequence may only register once.
• The genome whisperers downgraded the predicted danger of some gene variants from previous reports. A mutation can be more deadly in one population than in another – such as the BRCA genes. And they didn’t always agree on whether a particular gene variant would cause disease, nor about which findings should go on to the doc team.

WHAT DID PARTICIPANTS LEARN?
Each one had 100 or so “novel and rare genetic variants,” and 1 to 7 “personal disease-risk findings” that could, theoretically, harm health. I hate to say it, but they probably could have gotten some of this info from 23andme’s exome sequencing before the FDA silenced them, which went for $99 a few years ago.

Of the dozen participants, only one got an “actionable” report – she had a BRCA mutation and had surgery after learning of the result, which was very unexpected because she had no family history of cancer.

Brazilian_cifrano1 (1)What did all this info cost per patient? For sequencing plus interpretation about $15,000, plus another $1,000 for initial follow-up consults and tests, which sounds a little too rosy.

Whether that price tag is ultimately cost-effective or not of course depends on circumstance. If it identifies a rare disease, avoiding perhaps years of testing, then yes. But if it turns up a tendency to clot due to inheriting factor V Leiden, a genetic counselor could have caught that with an informative family health history and tests for mutations in blood clotting genes.

A PROBLEM THAT WILL VANISH WITH TIME
I’m not surprised that looking at the genomes of a dozen healthy people didn’t provide a crystal ball to predict their medical futures for a simple reason. The human genome is so complex, with instructions buried in layers of molecular language, that the very idea of going from sequence to diagnosis may be flawed, at least until we can work out all possible gene-gene interactions, against the backdrop of the environment. But this limitation is itself limited. It will go away with time, as more and more human genomes are subjected to the sequencers and the annotators, who then whisper to the clinicians what, exactly, to impart to a patient.

dnaAnd that’s why I will, one day, have my genome sequenced. But I’ll do it anonymously, so that my personal collection of variants can be considered along with everyone else’s to better inform clinicians on what hidden future illnesses their patients might bring with them to the exam room. If everyone does it, perhaps we won’t have to worry about privacy, for we all have genomic glitches. Decades ago geneticists called this fact “genetic load” — we all have our mutations. Now we can identify the glitches.

But even when we have complete genome sequences for millions of us, something I predict will be true within five years, genotype will not always predict phenotype. For DNA is not destiny.

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Mitohype: 3-Parent Designer Babies Who Will Change Human Evolution

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Mitochondria have their own genomes, but the nuclear genome dwarfs it.

Mitochondria have their own genomes, but the nuclear genome dwarfs it.

If I turned in a 20,337 word article and the editor decided to replace 37 of those words, would I call her a co-author? Certainly not. So why does replacing 37 genes in a fertilized ovum destined to develop into a sick child conjure up images of ménages-a-trois in Petri dishes and mingling chromosomes? Those genes, most of which control energy metabolism, are delivered in mitochondria that replace their mutation-bearing counterparts.

To read some of the media coverage a week ago, you’d think that the February 25th meeting of the  Cellular, Tissue, and Gene Therapies Advisory Committee at the Food and Drug Administration (FDA) was to discuss creating monsters, not manipulating mitochondria. But it’s not really the media’s fault. One of the researchers who spoke at the session recently published a technical article entitled “Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases,” so I suppose some mitohype is to be expected.

257px-Mad_scientist.svgETHICAL OBJECTIONS
I listened to the first day of the FDA meeting on assignment for Medscape. The focus was the state of the science that might or might not support approval of a phase 1 safety clinical trial of mitochondrial replacement. Several experts told of their preclinical work, on human cells and embryos of mice, cows, and monkeys, and the day wrapped with discussion by the FDA committee, which includes clinicians, scientists, and bioethicists. But in between was a session of public comment.

Two approaches are being considered: introducing the male and female nuclei from a fertilized ovum into an egg from a healthy donor that presumably contains healthy mitochondria, and “spindle transfer,” which uses the apparatus that divides chromosomes to deliver the mitochondria that naturally gather around it to power cell division. (Recall from 10th grade biology that a mitochondrion is “the powerhouse of the cell.”)

I found it odd that bioethics wasn’t on the FDA’s agenda, but thought perhaps the technology would be deemed too soon or off limits based on the science alone. I expected that the public commentators might be from families with mitochondrial diseases, but to my surprise the 7 speakers focused on bioethics.

A statement earlier in the month from the Center for Genetics and Society that appeared, in part, in the previous Sunday New York Times as “Genetically Modified Babies” set the tone. While I agree with the statement’s conclusions that “more than 250 signatories” endorsed that the technology simply isn’t necessary, it included the sort of inflammatory language that has plagued the modern biotech industry from the start in the 1970s. Listening to the public comments, after a morning of experimental details involving spindles, mitochondria, and pronuclei, the switch to research described as “authorized intentional genetic modification of children and their descendants” might have been literally accurate, but I found it jolting and seemingly missing the intent and extent of the technology.

Although some of the public comments addressed mitochondrial manipulation, for the most part they veered down that oft-evoked slippery slope to clones and bizarre vegetables. Here are a few:

“Who is the mother? One cell is just getting a couple dozen mitochondrial genes, but the woman’s egg that is enucleated is getting 20,000 genes. The new individual is the product of a massive procedure, like a genetically modified tomato. This new individual is a genetically modified human being.” (Hank Greely’s “Heather Has Three Parents” at the Law and Biosciences Blog from Stanford Law School points out that our genomes are modified, quite naturally, all the time.)

“It is a gateway technology to use SCNT (somatic cell nuclear transfer) or other methods in human trials. We need to look more carefully at animal research on cloning.” (The FDA discussion was on manipulating oocytes, not somatic cells. But cloning was mentioned so often that I was reminded of the old marijuana-leads-to-heroin argument. And nuclear transfer has been around since the 1960s, to clone non-human animals.)

“This biotechnology could alter the human species.” (Health care routinely alters evolution of our species.)

One otherwise eloquent speaker uttered the following so fast I could barely keep up. She had “Grave concerns” about the “creation of GM children,” “perversion of the relationship between parents and children,” “alteration of the human species,” and “GATTACA-like classes of human beings and the dissolution of our humanity.”

And finally, “we all remember Jesse Gelsinger,” said a prominent speaker gravely. Indeed we do. Jesse was 18 years old when he died following a gene therapy procedure, as I discuss in depth in my book on that biotechnology. Although Jesse Gelsinger was once a fertilized egg, he was never an oocyte, the subject of discussion. I think the point was informed consent.

The emotion and hyperbole perhaps weren’t necessary. As more than one committee member pointed out, it was an astute FDA scientist (Frances Oldham Kelsey, MD) who averted a thalidomide disaster in the U.S. back in the early 1960s.

As we await further public comment, possible until May 9, here’s some interesting facts about mitochondria that didn’t make it into most news coverage.

COOL THINGS ABOUT MITOCHONDRIA
Gerald Shadel, PhD, director of pathology research at the Yale School of Medicine, delightfully opened the morning session introducing mitochondria as “double-membraned submarines that cruise around cells but are actually very complex, forming large elaborate dynamic networks.” The biology of these cell parts is highly unusual, and that’s perhaps why the public discussion kept returning to the more familiar cloning. But a mitochondrion has nothing much in common with a cloned somatic cell.

Ancestral complex cells swallowed simpler cells, which became mitochondria, much like big fish swallowing little fish.

Ancestral complex cells swallowed simpler cells, which became mitochondria, like big fish swallowing little fish.

THE ENDOSYMBIONT THEORY Mitochondria look like bacteria, reproduce like bacteria by growing and splitting, and have their own DNA like bacteria. That’s because they likely descended from bacteria that were presumably swallowed up by the earliest complex cells. Today mitochondria are integrated parts of ourselves — the energy reactions that they house also require expression of genes from the nucleus.

MATERNAL INHERITANCE Eggs are packed with lots of stuff, including many mitochondria. Not so the streamlined sperm, whose mitochondria cling to its midpiece section, ready to fuel the  long swim to the egg. Should an errant mitochondrion sneak into a sperm head and survive the cervical journey and make it into an egg, maternal molecules soon dismantle it. Meanwhile, most of the 1 in 200 eggs that have a mitochondrial mutation stop developing. Isn’t the female body amazing?

The enzymes that carry out cellular respiration are arrayed along the infoldings of the inner mitochondrial membrane, in the order in which they are deployed. (Maureen Heaster)

The enzymes that carry out cellular respiration are arrayed along the infoldings of the inner mitochondrial membrane, in the order in which they are deployed. (Maureen Heaster)

UNPREDICTABLE VARIABILITY Body (somatic) cells have 2 copies of each chromosome, and therefore 2 copies of each gene. But cells have many mitochondria, especially skeletal muscle cells, which can have thousands. That’s why a mitochondrial disease often causes great fatigue and weakness.

A mitochondrion has several copies of its tiny genome, each one a mere 16,569 DNA nucleotides, compared to the 3 billion or so in the nucleus. The genetic landscape of the mitochondria in a cell is more a population of gene variants than the 1:1 ratio seen in a person who is a carrier (heterozygote) of a nuclear gene.

If a woman is a heterozygote for a mitochondrial gene (has two variants), as the number of mitochondria whittle down from 100,000 to about 100 as the egg matures, some eggs end up with about equal copies of each gene variant, but most are skewed, getting all healthy versions or all bad ones. This unpredictable inequality, called heteroplasmy, means that a woman can be healthy, but have a child with a mitochondrial disease when the developing egg unluckily picks up many copies of a mutation.

Heteroplasmy also means that siblings may be affected to very different degrees, that symptoms may not start until enough cells with mutant mitochondria accumulate, and that mitochondria in one cell type may be packed with the mutation but not so others, complicating diagnosis based on symptoms and testing an accessible body fluid.

Heteroplasmy complicated forensic identification of Tsar Nicholas II and his family. (Armed Forces DNA Identification Lab)

Heteroplasmy complicated forensic identification of Tsar Nicholas II and his family. (Armed Forces DNA Identification Lab)

TSAR NICHOLAS II   On a July night in 1918, Tsar Nicholas II of Russia and his family, the royal Romanovs, were shot, their bodies damaged with acid and buried in a shallow grave. In July 1991, two amateur historians found the grave and sent DNA samples for testing. Y chromosomes distinguished the males and mitochondrial DNA (mtDNA) identified the Tsarina and her three daughters.

But probing the DNA of descendants of the royals showed that the remains thought to be the Tsar differed at base 16169 in the mtDNA from that of his living great-grandniece Xenia. The Tsar’s mtDNA had T at the site in some samples, C in others.

Before we knew much about the changeability of the mitochondrial genome – it doesn’t repair itself like nuclear DNA and is splashed with oxygen free radicals from all those energy reactions – forensics researchers thought the Tsar’s strange DNA must have been due to a sequencing error. But then in yet another July, in 1994, researchers exhumed the body of Nicholas’s brother, Grand Duke Georgij Romanov. His mtDNA at position 16169, in bone cells, also went both ways, with a T or a C. 

The heteroplasmy that confused forensic analysis of the Romanovs isn’t rare after all. Sequencing of many mitochondrial genomes has revealed that one in ten bases can differ within an individual.

Coenzyme Q, aka ubiquionone

Coenzyme Q, aka ubiquinone

COENZYME Q   This molecule that takes part in the reactions of cellular respiration graces the shelves of health food stores, and is in dozens of clinical trials to evaluate treatment of a wide range of neuromuscular disorders, heart disease, and reproductive uses. It’s in phase 3 clinical trials to treat mitochondrial diseases.

TROUBLESHOOTING
After the expert presentations and public comments, the FDA committee members, including scientists, physicians, and bioethicists, listed the science-based problems with mitochondrial manipulation that had emerged:

Will mixing mitochondria and eggs from two populations be a problem? Carlos Moraes, PhD. of the University of Miami Miller School of Medicine offered the example of a Brit going to Australia and marrying an aborigine to make the point that it wouldn’t.

Heteroplasmy. It happens, but in non-human animal studies hasn’t been a problem.

Carryover. How can we know if some mutation-bearing maternal mitochondria get into the manipulated fertilized ovum? If it does, will it affect health? Over time, heteroplasmy does tend to shift towards favoring one gene variant. The risk of carryover is unknown.

Could the delicate fertilized ova be damaged or lose chromosomes? Sure. That’s a risk of IVF, but preimplantation genetic diagnosis (checking a cell of an early embryo) can get around that.

Could resulting children be damaged? Possibly. IVF increases the risk of Beckwith-Wiedemann Syndrome, an overgrowth condition that predisposes to cancer. The link took years to show up because the condition hadn’t been seen in animal models.

(NHGRI)

(NHGRI)

Katharine Wenstrom MD, a clinical geneticist from the Alpert Medical School of Brown University, summed matters up. “A lot of patients don’t develop symptoms until adulthood because it takes that long for abnormal mitochondria to accumulate. This makes me nervous to talk about a healthy blastocyst being good to go, or an animal model. There are so many aspects of mitochondrial disease that we don’t understand, such as tissue specificity, changes over time, and response to environmental stimuli.”

PRACTICAL MATTERS
I heard several people mutter “adoption” during the late-afternoon discussion as an alternative to creating a fertilized ovum with healthy mitochondria. Using a donor egg is another option.

I’d wondered why members of families with mitochondrial disease hadn’t been among the public commenters. Then, at the wrap-up, Sharon Reeder eloquently and non-hysterically put everything into perspective.

“How can we prevent when we can hardly diagnose? I was diagnosed 14 years ago. It took 16 years. My first symptom, when I was 18, was a droopy eyelid. They fixed it, and nobody asked why. I had a child when I was 35 and when I was 36 I was diagnosed. Pregnancy and giving birth were incredibly hard. I ended up in a wheelchair after I gave birth. I was negative in blood but positive in a muscle biopsy. I now have 10 doctors. Healthy people don’t go in to get their mitochondria checked.

I’m sitting here thinking, ‘Oh gosh! It would be so great if I was listening to all this research and it was about therapies for those of us with mitochondrial disease, helping those of us whose lives are severely affected. But this might be the gateway to that.”

At the risk of misinterpreting Ms. Reeder, the gateway that she mentions differs from the gateway to the slippery slope that would lead from research on mitochondrial replacement to the making of designer 3-parent babies that would disrupt the parental-child bond and alter the course of human evolution forever. I think she means that even if this particular biotechnology never makes it to clinical trials – for whatever reasons – what we learn from the journey could ultimately translate into treatments based on new understanding of the tiny genomes within the still-mysterious powerhouses of the cell.

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Rare Disease Day 2014: A Parent Fights to Cure Blindness

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Kristin Smedley, fighting for her sons' vision.

Kristin Smedley, fighting for her sons’ vision.

For Rare Disease Day 2014 tomorrow, I’ve asked Kristin Smedley, president and co-founder of the Curing Retinal Blindness Foundation, to guest post. I introduced her here last year, Rare Disease Day: What 5 Kids With Low Vision Can Do. Kristin and Mike’s sons Michael and Mitchell have the CRB1 form of Leber congenital amaurosis. CRB1 is an acronym as well as the name of the errant gene.

The Internet has been absolutely wonderful in bringing together families, with the same or different genetic disorders, facing similar challenges — and conquering them.

Go Kristin!

Never doubt that a small group of thoughtful, committed citizens can change the world; indeed, it’s the only thing that ever has.” Margaret Mead

CRB1 degenerative retinal disease is a rare disease, affecting an estimated 300 people in the US. Yep, it is THAT rare. It causes visual impairment that eventually degenerates the retina to total blindness. Although some characteristics are similar, it is not as “popular” as macular degeneration, and isn’t prevalent in the “Baby Boomers” population — so you’ve likely not seen CRB1 in the headlines … yet.

February 28

February 28

A group of CRB1 families is working to change that, to literally put CRB1 in the headlines to get the attention it needs, to fund the science to treatments and cures. I lead this group, and in less than three years since we launched our effort, we’ve already hosted our first research symposium, formed a stellar scientific advisory board, funded our first 5 grants for CRB1 research worldwide, and have started locating CRB1 families all over the continental US and abroad. And we aren’t stopping. With a goal of multiple treatments for CRB1, we have work to do.

MOTIVATION
So how can parents not formally trained in genetics or ophthalmology carve out a path, multiple paths, to treatments and cures? For starters, motivation is key. When you are in the depths of blindness every single day with a child, it is easy to “see” where the motivation of the CRB1 families comes from. Take my family for instance.

I have two sons affected.

Two sons who want to be baseball pitchers.

Two sons who want to be quarterback.

Two sons who want to drive. But due to this rare disease, they can’t … at least not yet.

mitchell crb1 2014INSPIRATION
When my husband and I were finally given test results that confirmed our oldest son has the CRB1 genetic disease, we immediately started asking if there was something we could do to get research going. Circumstances led us to some miraculous connections:

We had the opportunity, the blessing, to meet some folks who had just opened the door to gene therapy for a genetic blindness like CRB1: Dr. Jean Bennett and Dr. Eric Pierce. And then I met families working to open the gene therapy door for their form of blindness like CRB1: Jennifer Pletcher of  Finley’s Fighters and Jennifer Stevens of Gavin’s Groupies. I even had the good fortune to connect with Ricki Lewis, author of The Forever Fix, and through her met the super-mom Lori Sames of Hannah’s Hope Fund. All of these connections gave us inspiration, and concrete ideas, for our CRB1 families to move, in a BIG way, to plan our own path to treatments and cures.
michael crb1 2014

COLLABORATION
Obviously parents are quite motivated, but motivation can only carry you so far. We need to progress quickly. Time is not on our side as our kids that still have vision are losing it every single day. We can’t afford to reinvent the wheel, or waste fundraising dollars. So we look for every opportunity to collaborate, to partner with others who have a resource we need or a means to a resource to get what we need.

We needed to build a Scientific Advisory Board to help us utilize every dollar efficiently and effectively, so we went to a conference dedicated to our sons’ umbrella disease, Leber Congenital Amaurosis, and approached every single presenter, and asked them to help.

We needed to establish a solid 501(c)3 non profit organization, so I contacted the highly regarded National Organization for Rare Disorders for guidance.

We needed to find out what government resources are available to us and what issues we should deal with now in preparation for the eventual clinical trials, so we traveled to Rare Disease Day events to connect with the National Institutes for Health and other fantastic organizations.

We needed to connect with the best researchers in the world to invite them to join our mission and help move the science forward, so we attended to the  Association for Research in Vision and Ophthalmology annual meeting to connect with brilliant scientists. And now, as we are looking to find as many CRB1 patients as we can, we are proud to be working with the Foundation Fighting Blindness to establish a CRB1 patient registry.

cure-retinal-blindness-foundationDEDICATION
Since we utilize every opportunity to network and gather resources, it’s not surprising that our fundraising is growing and our outreach is spreading every day. Research teams are now contacting us to help. And last week within a 7 day span, I was contacted by families in Puerto Rico, Belgium, United Kingdom, and Mexico, all of whom have received the devastating diagnosis of CRB1 retinal disease and are looking for support … and more.

Just like me 14 years ago, when I got the first of two diagnoses, these families are looking for Hope. There was no Hope just over a decade ago, but we created it.

Families now have Hope when they connect with others with the same disease.

Families now have Hope when they are able to read through a list of descriptions of research being conducted on this disease.

Families now have Hope knowing that our group is dedicated, and our scientists are dedicated, and our supporters are dedicated to SEEing this all the way through to the cure.

Alone we can do so little. Together we can do so much.” Helen Keller

Read Kristin’s blog “Eye Believe in Miracles”

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Exon Skipping: Borrowing from Nature to Treat Rare Genetic Diseases

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1000px-Skipping_Stones.svg Imagine selecting, copying, and pasting this sentence into a new document, but dropping or adding a word. The meaning might change — in the copy.

The same thing – gaining or losing information – can happen to a gene, because like sentences built of words, most genes come in pieces. For decades researchers have been trying to co-opt nature’s way of copying only some of a gene’s information into messenger RNA (mRNA) to bypass harmful mutations as if they are typos. The strategy, called exon skipping, is finally nearing the clinic.

GENES IN PIECES
Understanding exon skipping requires a short detour into the biology.

(Laurence Hurst et al, PLOS Biology)

(Laurence Hurst et al, PLOS Biology)

I was in grad school in February 1978 when Harvard’s Walter Gilbert published a short News & Views piece in Nature –  “Why Genes in Pieces?”, that changed what everyone thought they knew. A gene’s information isn’t continuous, but is dispersed in exons alternating with seemingly meaningless introns.

Before an mRNA is translated into protein, enzymes snip out its introns at splice sites. If a mutation alters a splice site, results can be catastrophic. The mRNA can retain an intron or lose an exon, like a sentence with an extra or missing word, and the protein product is too long or too short. Some genes are normally spliced in different patterns, like creating different sentences from one long one, leading to versions of a protein adapted to different circumstances.

In the budding biotechnology of exon skipping, splice sites are chemically shielded in ways that enable an mRNA to form while ignoring a mutation, or altering how the RNA or protein folds. To do that, an “antisense” molecule binds to a specific sequence in the mRNA like a Velcro patch to cloth. The binding is based on the complementary base pairing (A with T or U, G with C) of nucleic acids. If a stretch of RNA is GCUA, the corresponding antisense sequence would mimic CGAU.

ALMOST READY FOR PRIME TIME

February 28

February 28

Antisense silencing of mRNAs isn’t new, and began in agriculture. I wrote “Building a Better Tomato” for High Technology magazine in 1985, and “Making Sense of Antisense” for BioScience in 1989. But this month’s special issue of Nucleic Acid Therapeutics, on exon skipping, indicates that the field has gone far beyond slow-ripening tomatoes.

A Guest Editorial by Annemieke Aartsma-Rus. PhD, in the Department of Human Genetics, Leiden University Medical Center, introduces the European Cooperation in Science and Technology (COST), with an action mandate to “overcome challenges through networking to allow clinical implementation of antisense-mediated exon skipping for as many rare disease patients as possible.” Just in time for Rare Disease Day next week.

COST will sponsor private workshops to discuss preclinical results, try to lower regulatory hurdles for testing treatments for extremely rare variants of rare diseases, standardize testing of the proteins that indicate whether exon skipping is working, and identify biomarkers from accessible body fluids to replace painful muscle biopsies and limited brain imaging.

(Dept. of Energy)

(Dept. of Energy)

The list of disease candidates for the technology is growing. Duchenne muscular dystrophy (DMD) is farthest along, with a phase 3 clinical trial just completed. Leiden University’s Dwi Kemaladewi shows how it works in this video, a winner of Science magazine’s “Dance Your PhD” contest from 2011. Other targets are a rare form of Alzheimer’s disease, spinal muscular atrophy, Leber congenital amaurosis (blindness) due to CEP290 mutation, and fibrodysplasia ossificans progressiva, which turns muscle to bone.

Three diseases represent the past, present, and future of exon-skipping: familial dysautonomia, DMD, and Huntington disease.

FAMILIAL DYSAUTONOMIA
I first heard about exon skipping in the context of the very rare familial dysautonomia (FD), where it occurs naturally. FD is one of the “Jewish” genetic diseases – my friend’s daughter has it. The single-base mutation is at a splice site between the 20th intron and exon pair of a gene called IKBKAP.

FD affects neurons that control such autonomic functions as breathing, digesting, regulating temperature, sensory perception, and making tears. Some symptoms ebb and flow with crises of shaking cold and pervasive nausea and dizziness, while others, such as the need for tube feeding, are a constant for some people. A character in a 2010 novel by Lionel Shriver, So Much For That, shows what life can be like with FD.

But FD has a peculiarity that inspired the idea to exploit natural gene splicing– some cells ignore the mutation. They manufacture a normal protein, possible because the amino acid sequence information is still there, it’s just the splice site that the mutation alters. It’s a little like a lit indicator on a car’s dashboard signaling a problem that isn’t really there.

If cells that ignore the mutation and make IKBKAP protein happen to be in the parts of the brain that the disease affects, the child is lucky and may have a mild case – like my friend’s daughter, who tells her story in my human genetics textbook.
.
For FD patients not so lucky, cells from the brain and spinal cord skip the exon, while muscle, lung, liver, white blood cells, and glands – parts unaffected in the illness — produce normal-length proteins.

Fukuju_green_tea_leavesIn 2003, discovery of the FD mutation and the tendency of some cells to ignore it inspired Drs. Berish Rubin, Sylvia Anderson, and their colleagues at Fordham University to screen compounds that might modulate levels of the protein, perhaps by affecting splicing. They found two –- a form of vitamin E (tocotrienol) and a green tea component (epigallocatechin gallate), and another team last year added phosphatidylserine. (I’m not a doc, so for info on these supplements, see FD Now.)

DMD – LEADER OF THE PACK
In FD, a skipped exon in brain cells causes the symptoms. In DMD, inducing exon skipping helps. Dutch biotech company Prosensa, with GlaxoSmithKline, has just completed a phase 3 clinical trial of drisapersen and Sarepta Therapeutics is nearly as far along in testing its candidate, eteplirsen The two approaches use different chemistries to create their “antisense oligonucleotides” (AONs), which bind specific splice sites.

The clinical trials have had recent ups and downs, mostly because tests of efficacy must go on for a long time to demonstrate that an intervention is actually having an effect, and that apparent progress isn’t just due to natural fluctuation of symptoms. A longer-term goal is to figure out a way to deliver the treatment to enough muscles to improve mobility and quality of life.

Despite these hurdles, the therapy itself is sheer genius.

In DMD, fat gradually replaces muscle.

In DMD, fat, the white blobs in the lower muscle cell crossection, gradually replaces muscle.

In some boys with DMD, a mutation in exon 50 of this gargantuan 79-exon gene introduces a nonsense mutation, which is like a period appearing in the middle of a sentence, truncating it. The encoded protein, dystrophin, is too short to do its job of supporting a muscle cell membrane during a contraction.

But in the much milder Becker muscular dystrophy, a less disruptive mutation yields a slightly shortened but partially active dystrophin. Lifespan is normal. This means that some restoration of dystrophin function can, theoretically, perhaps help a boy with DMD, who faces a very limited future.

Indeed, when AONs bind near the nonsense mutation that causes DMD, the “spliceosome” that does the cutting dances right past the glitch and “restores the reading frame” (the sequence of base triplets that encode the amino acids). Some dystrophin is made. The effect is a little like ignoring a series of miskeys in a typed sentence.

But does the fix at the molecular level improve the performance of boys with DMD in a 6-minute test of walking ability? How far a boy can walk in this time may seem an arbitrary sort of measure, but it’s based on years of research into understanding the “natural history” of the disease. By identifying exactly which functions ebb and at what rate, the natural history provides the benchmarks against which a new treatment is assessed.

For DMD, the natural history is a sad series of losses. Craig McDonald, MD, a professor of pediatrics at the UC Davis School of Medicine, described it in a news conference Prosensa held in late 2013. “The time to stand predicts time to loss of standing, and loss of standing predicts the age at loss of ambulation. Age at loss ambulation predicts age at loss of self-feeding and need for assistance in ventilation. Inability to jump, hop, run, standing from the floor or form a chair, stair climbing, even the ability to stand in place, reach overhead, or put hands on a tabletop, are meaningful ambulatory milestones.”

The goal of the drugs that manipulate gene splicing is to slow this relentless course. And that’s why the observation that boys under age 7 given drisapersen for 96 weeks walked on average 49 meters more than those on placebo was good news. But the FDA sent the other drug back for further testing, because the 6-minute walk test is so variable that more time is required to see if the improvement persists. So stay tuned – both drug candidates seem well on the road to approval.

The yellow blobs are inclusions of misfolded Htt protein in this section from an HD brain.

The yellow blobs are inclusions of misfolded Htt protein in this section from an HD brain. (Steven Finkbeiner)

HUNTINGTON DISEASE
If FD provides exon skipping’s basis and DMD becomes first to hit the clinic, then maybe HD represents future success. A recent DNA Science post described the tragedy of HD in one young family.

HD is a classic “expanding triplet repeat” mutation — affected individuals have extra copies of the sequence CAG in exon 1 of the gene that encodes the protein huntingtin (Htt). The extra stuff tacked onto one end of the gene distorts how its mRNA folds, affecting splicing in a way that generates “toxic fragments” of protein.

Melvin Evers and co-workers, also from Leiden University, report in February’s Nucleic Acid Therapeutics how they deduced that skipping exon 12 (out of a total 67) of the HD gene might prevent release of the toxic fragments. So far the approach has worked in HD patients’ cells and in a mouse model of the disease. Because HD is a disorder of extra genetic material and currently has no treatment, gene silencing seems a logical approach.

RICKI’S WRAP-UP
The story of the harnessing of exon skipping provides a great example of the evolution of science to technology. From 1978’s “Why Genes in Pieces?” to little boys with muscular dystrophy gaining steps has taken 36 years. In the interim lay much hypothesizing about why most genes are patchworks. Now that we know that what was once thought to be exception is actually the norm, we can perhaps put mosaic genes in the context of being another manifestation of genome versatility.

The long, slow gestation of exon skipping as a therapeutic strategy stunningly counters the BREAKTHROUGHidea, still propagated in the news media, of the overnight medical breakthrough.

Insight and application take time.

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Patient-Specific Stem Cells Recapitulate Age-Related Macular Degeneration

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Age-related macular degeneration obliterates the visual field, from the center outwards. (Natl Eye Inst)

Age-related macular degeneration obliterates the visual field, from the center outwards. (Natl Eye Inst)

Stem cell debate and hype continue, with each advance distancing the field from embryos while promising replacement parts as stem cells “turn into” everything from hearts to gizzards. Meanwhile, many researchers have been quietly pursuing the immediate promise of the cells – conjuring the beginnings of diseases in dishes. DNA Science featured “brain organoids” last summer, before the world disappeared beneath a glacier.

iPS CELLS TAKE ON THE EYE
Induced pluripotent stem (iPS) cells, more commonly called reprogrammed cells, start out as skin or other somatic (body) cells, zip back in developmental time to a stem-cell-like state, then are coaxed to assume whatever guise researchers wish to study. The cells are the route to personalized implants, because they come from the patient who needs a spare part. But that will require a lot of testing. More immediate, and more exciting I think, is when iPS cells serve as living time machines.

Imagine taking an affected cell from a person very sick from a degenerative disease, and reversing the clock, glimpsing in a lab dish how things began to go wrong.

A good disease to dissect using reprogrammed cells is age-related macular degeneration (AMD), a form of encroaching blindness that affects 12 to 15 million people in the U.S. and a quarter of those over 60. Incidence will double as the population ages over the next decade. In AMD the visual field becomes wavy, faded, and blocked, from the center outwards, greatly interfering with daily life.

iPS cells (Stephen Tsang and colleagues, Columbia University)

iPS cells (Stephen Tsang and colleagues, Columbia University)

AMD is a great candidate to mirror in reprogrammed cells because iPS cells left alone and not given biochemicals to steer their specialization will, for reasons unknown, eventually become the very tissue responsible for the gradual visual loss – the retinal pigment epithelium, or RPE. A DNA Science post from last year describes the RPE in detail.

THE ANTI-OXIDANT CONNECTION
Like many diseases, AMD arises from an interplay of environmental and genetic influences. A team led by Stephen Tsang, an ophthalmologist and geneticist at the Harkness Eye Institute at Columbia University, used iPS cells to reveal the gene-environment interaction that underlies AMD, with a practical result for patients. Their report appeared in the February 4 Human Molecular Genetics.

CarrotsIn both the “dry” and “wet” forms of AMD, the body is less able to temper formation of “reactive oxygen species,” molecules that fling off extra energy that damages cell parts. The enzyme superoxide dismutase 2 (SOD2), made in mitochondria in many cell types, normally supplies this antioxidant activity, but it’s deficient in some people. The National Eye Institute’s AREDS (Age-Related Eye Disease Study) recommended that patients at high risk for AMD take certain antioxidants (vitamins C and E and beta-carotene) plus zinc and copper.

The major risk factor for developing AMD, besides the “age” in the disease’s name, is smoking. Variants of three genes contribute to the risk too, but to a lesser degree. These variants combine to provide a “risk” genotype that increases chances of AMD, and a “protective” genotype. Seeing how iPS cells expressing the two genotypes differ would provide a window into the gene-environment connection, while indicating which patients can actually benefit from taking the recommended antioxidants.

WATCHING AMD IN VITRO
The RPE, a thin layer that hugs the photoreceptors (rods and cones), is a garbage dump of sorts for broken down pigments that can generate reactive oxygen species. The rods and cones continually shed pigment-rich pieces of themselves as they break down vitamin A whilst transducing photon energy into signals to the brain.

Over time, yellowish-brown “aging” specks, collectively called lipofuscin pigments, come to pepper the cells of the RPE. These are the same dreaded “liver spots” that appear on skin as we age. One type of pigment in the RPE is called A2E. Expose it to blue light, and reactive oxygen species form.

To recapitulate AMD, the researchers created iPS cells from skin fibroblasts from four patients: 2 controls without AMD, one with two copies of the risk genotype, and a fourth participant with one protective and one risk genotype.

Speckled cells appearing in the sheet of iPS cells are RPE. (Stephen Tsang and colleagues, Columbia University)

Speckled cells appearing in the sheet of iPS cells are RPE. (Stephen Tsang and colleagues, Columbia University)

Two to three months later, RPE cells emerged from the sheets of iPS cells, appearing as a cobblestone-like pattern of pigmented cells resembling bathroom tile.

Next, to simulate aging, the researchers added A2E and blue light for 10 days to the cells, dubbed iPSC-RPE. Watching the sped-up aging showed the pathology in a way that isn’t possible probing the damaged RPEs in eyes from eye banks that have stripped off the corneas or from autopsies on long-blind patients. Those RPEs, if they are there at all, are typically shredded into uselessness.

A battery of tests chronicled the iPSC-RPE cells aging. Microscopy showed lipofuscin pigments accumulating, and mass spectrometry revealed the spectrum of proteins in the cells from the 3 types of patients – fully protective genotype and healthy, and fully at-risk and the hybrid.

Superoxide dismutase (SOD2), our antioxidant enzyme

Superoxide dismutase (SOD2), our antioxidant enzyme

The cells from the two controls poured out SOD2 after A2E exposure, explaining why those two individuals don’t have AMD. But the cells from the AMD patients made only negligible amounts of the antioxidant enzyme. And that distinction creates a biology-based, personalized approach to taking supplements. “Instead of giving AREDS cocktails, we can now do a skin biopsy and then give antioxidants only to those who have poor SOD2 responses,” Dr. Tsang told me.

WHY THIS STUDY IS COOL
1. It’s a great example of personalized medicine – with the caveat that antioxidants protect against conditions other than AMD.

2. It validates a technique that I once harbored qualms about: genome-wide association studies (GWAS).

The awkwardly-named method identifies parts of the genome that people with a particular trait or illness share, pointing to regions where causative genes may lie. Early on, GWAS results were sometimes retracted after adding data dispelled the associations. And many GWAS identified gene variants that contribute only tiny degrees to a trait. But more recently, as the numbers have grown (participants profiled and genome regions probed), GWAS have indeed led to identifying genes of interest.

The functions of the three genes considered in this study (CFH, ARMS2 and HTRA1) found using GWAS, weren’t known. But the iPSC-RPE cells clearly demonstrated their role in the antioxidant response – when mutant, cells of the RPE can’t handle the oxidative stress of accumulating aging pigments.

3. The study elegantly shows how the cell bridges the molecular and the medical. Clinical researchers can study a disease in people, geneticists can sequence the underlying faulty instructions, and molecular biologists can decipher the biochemical pathways that detour, causing disease. But watching that disease unfold in cells really reveals the pathology.

Reprogrammed, patient-derived, iPS cells provide that priceless peek – with nary an embryo in sight.

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Probing Polar Bodies to Pick Disease-Free Embryos

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A polar body is to an oocyte what a moon is to a planet. (NASA)

A polar body is to an oocyte what a moon is to a planet. (NASA)

After writing eleven editions of a human genetics textbook, I automatically assign chapter numbers to exciting new findings. But the 3-page case report in this week’s JAMA Neurology on selecting disease-free embryos tangled up my brain with all its connections.

The case report delves into:
a. Meiosis, including polar bodies
b. Mendel’s first law
c. Embryology
d. Prions
e. Protein folding
f. Assisted reproductive technologies (ARTs)
g. Neurogenetics
h. Bioethics
i. All of the above

The researchers (Alice Uflacker and Murali Doraiswamy from the Duke Institute for Brain Science, Svetlana Rechitsky and Ilan Tur-Kaspa from the Reproductive Genetics Institute in Chicago, and Michael Geschwind and Tricia See from UCSF) describe using ARTs to enable a woman whose relatives have a devastating and very rare brain disease to have children free of the family legacy.

AN INHERITED PRION DISEASE
The disease, Gerstmann-Straussler-Scheinker Syndrome (GSS), affects only 1-10 per 100 million births. Much of what’s known about it comes from an 8-generation family from Indiana that has had 57 affected members.

GSS is a prion disorder, but one that is inherited rather than acquired from eating tainted burgers from mad cows (bovine spongiform encephalopathy) or the brains of dead people  (kuru). A prion is a protein that can exist in several folded forms, one of which is infectious – it makes the other forms like itself. These “rogue proteins“ tend to turn brains into a spongy mess.

Cannibals, Papua New Guinea

Cannibals, Papua New Guinea

Prions were first described in 1954 in sheep afflicted with scrapie, but farmers had reported it since the Middle Ages. Chronicling of kuru among the Fore people of Papua New Guinea was the life’s work of D. Carleton Gajdusek, from the 1950s for many years before his legal woes. I tell the curious history of prions in chapter 4 of my essay book Discovery: Windows on the Life Sciences (Blackwell Science). (It was poorly marketed into oblivion.)

Women and children Fore infected themselves when they ate the raw brains of dead friends and relatives to honor them. The men got safe cooked parts, mostly muscle, and wives of dead warriors ate the penises, cooked I presume. You can read the gory story in Dr. Gajdusek’s Nobel speech. Then in the 1980s came the notorious stumbling bovines of England.

Because a prion is a protein, it’s encoded by a gene — the prion protein gene (PrP) on chromosome 20. Mutations at different places in the gene cause different inherited prion disorders, all of which are exceedingly rare.

Mad_cowCreutzfeldt-Jakob disease (CJD) is like mad cow. Fatal familial insomnia (FFI) is an inherited prion disease that may have inspired an illness among the crew of the USS Enterprise in Star Trek: The Next Generation (“Night Terrors,” which aired March 16, 1991, Stardate 19144631.2) The ship’s wandering into a rift in space deprived crew members of dream sleep, causing terrifying hallucinations and extreme paranoia. But unlike Commander Riker and colleagues, people with FFI do not sleep at all. Nor do they recover at the end of the episode.

GSS progresses from memory loss and slurred speech to “prion dementia,” uncontrollable movements, limb weakness, and sometimes deafness and/or blindness. Gummy prion protein is deposited in the cerebral cortex, the basal ganglia, and especially in the cerebellum, which destroys voluntary movement.

Alice Uflacker, MD, one of the Duke researchers, describes the condition. “Typical age of onset is the 40s to 50s. Disease progression is longer than that of genetic CJD and FFI. A patient may be symptomatic for about 5 years, leading to death. Because age of onset is past young reproductive age, patients may not be aware that there is a 50% chance of passing the mutation to their offspring. Often times, however, the person at risk has contact with immediate and extended family members and has witnessed their loved ones deteriorate.”

The patient, Amanda Kalinsky, and her husband and children appeared on the front page of the New York Times on Tuesday.

February 28

February 28

Because GSS is autosomal dominant, it peppers family pedigrees in each generation, striking men and women. Even people who know their family history may have difficulty finding a physician who has heard of GSS, a problem that unites the rare disease community. Orthopedic surgeons, the specialists usually consulted when symptoms begin, look for common causes (“horses”) rather than the rare ”zebras.”

Many physicians also haven’t heard of preimplantation genetic diagnosis (PGD), although it’s been around awhile. The Kalinskys learned about it from a genetic counselor, and elected to have predictive testing so Amanda could learn whether or not her father’s disease lay in her own future. It was a brave decision that few in her position for similar conditions, such as  Huntington Disease, make. Now she knows she’ll develop GSS, for the disease has near-complete penetrance – inherit the mutation and you get the disease.

Amanda and her husband didn’t want her genetic fate for their children. And thanks to technology, they had a choice.

Embryo,_8_cells,_transparent_imageTESTING EMBRYOS
Selecting embryos isn’t new – it was first done in 1990 for X-linked mutations. In 1993 researchers selected the embryo that became Chloe O’Brien, free of the severe cystic fibrosis that affected her brother. In 1994 came another milestone, a girl conceived and selected to provide umbilical cord stem cells to treat her teenage sister’s leukemia, echoed in Jodi Picoult’s novel My Sister’s Keeper. The most famous PGDer was Adam Nash, selected to cure his sister of Fanconi anemia and born in August 2000. I vividly remember the negative vibes against this first “savior sibling” family on the Today Show; now the choice to have one child to help another isn’t so unusual.

PGD works because of a feature of the early embryos of many animal species called indeterminate cleavage. A cell can be plucked from an 8-celled embryo, tested, and the 7-celled remainder put back into a woman to continue developing, or held over for a few cell divisions. If the 7-celled embryo has the probed mutation, it can be discarded or used in research to study the genesis of the family’s disease. Some people who consider life to begin at conception object to the fate of the unused embryos. But thousands of children have been born without their family’s genetic disease thanks to PGD.

To circumvent objections to testing 8-celled embryos, researchers can use a technique called “sequential polar body analysis.” Polar bodies are by-products of egg formation that are Nature’s way to pack nutrients and organelles into a gigantic egg, prepping it to support an early embryo. The World Health Organization first suggested genetic testing of polar bodies to infer the genotype of the egg in the early 1980s. The intervention destroys the polar bodies, but they serve no function once they’ve  siphoned off extra genomes and built up the egg. They’re expendable.

CHROMOSOME CHOREOGRAPHY
Although both sperm and eggs carry only one copy of the genome so fertilization can restore the double number, their timetables are markedly different. Sperm develop quickly and equally. That’s not the case for the female cells.

Technically, the female cell is called an oocyte until it’s a fertilized ovum, so there’s really no such thing as a lone ovum. Oocytes jettison parts of themselves as they form by a double division (meiosis), yielding one huge oocyte and three much smaller cells, the polar bodies. Each of these four cells houses a single genome. In fact, researchers from Harvard and Peking University have already sequenced polar body genomes to infer the genome sequence of the oocytes  to which they cling. The name “polar body” is celestial and not ursine, because a polar body is like a moon that travels along with its planet.

The "polar" in polar body is a celestial reference, not an ursine one.

The “polar” in polar body is a celestial reference, not an ursine one.

The jettisoned polar bodies hold important clues, because as chromosome pairs part, a mutation that ends up in a polar body doesn’t end up in the all-important oocyte, or vice versa. This is the physical basis of Gregor Mendel’s observation of the segregation of traits in pea plants. So researchers can test the genes of a polar body to deduce which gene variants made it into the oocyte – in the case of Amanda Kalinsky, the GSS mutation or the normal version of the gene.

The researchers looked at the prion protein gene and 5 markers bracketing it on chromosome 20 in the polar bodies hanging onto some of Amanda’s retrieved oocytes. A different set of markers accompanied the mutant PrP gene and its normal (“wild type”) version, so they could be distinguished.
85331_large

But there’s more. Female meiosis actually spawns two sets of polar bodies, at each of the two stages of the division. Examining the markers of the later-released polar bodies can reveal whether the genes on the chromosomes swap parts, called crossing over. If so, then a false negative or false positive oocyte choice could result. A paper from 2011 from Anver Kuliev and Svetlana Rechitsky (who is on the JAMA Neurology paper) reports polar body testing for 938 cycles for 146 different single-gene diseases, resulting in 345 healthy children. It works. But it isn’t really a way to avoid intervening in prenatal development.

Let’s return to the issue of timing. The first meiotic division happens when an oocyte pops out of an ovary sometime after puberty. But the second meiotic division occurs as fertilization happens. So probing polar bodies to catch those confusing crossovers, while a valid and earlier substitute for the 8-cell-stage PGD, happens at the exact time of fertilization. And as I know well from the personal name-calling that followed my recent DNA science post When Does a Human Life Begin? 17 Timepoints, many people do consider  a merged sperm and egg to be a full-fledged person, equivalent to say a 53-year-old accountant. Shifting the time of selection to the very beginning of development, to a single cell, might not make a difference to them.

Anyway, the polar body technique, validated with 8-cell-stage PGD, served the Kalinskys well. The researchers injected sperm into 14 oocytes (a refinement of IVF called ICSI, for intracytoplasmic sperm injection), lost a few along the way, but the polar body testing indeed revealed a crossover event that could have led to a mistake. Three beautiful children free of GSS ultimately resulted – 3-year-old twins and a baby, shown on the front page of the New York Times.

64px-Question_mark_alternate.svgTHE TOUGH QUESTIONS
The media focused much more on gathering bioethicists for comments than on explaining polar body biology or prions, so I’ll just touch on those issues, since I teach “genethics”:

Should an embryo be rejected because it has inherited a disease that won’t cause symptoms for half a century? That question has been raised for the BRCA genes, even more controversial because they confer susceptibility to treatable conditions.

The slippery slope. Will we slide from preventing future people from having deadly brain diseases to choosing trivial traits? We already have. PGD is misused in sex selection.

Are all these manipulations eugenic? Not by intent, but perhaps in consequence. Eugenics has a societal goal, and can be negative (kill the imperfect) or positive (reward the perceived best for reproducing). Medical genetics aims to alleviate suffering at the individual and family levels, but some interventions will ultimately affect the gene pool.

This landmark report on the rarest-of-the-rare Gerstmann–Sträussler–Scheinker syndrome, a unicorn among the zebras, provides some assurance that for those electing to choose embryos that have won the genetic roulette and not inherited the family’s disease, results are reliable, and can be done before the fertilized ovum divides.

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Project Prakash: Learning From the Formerly Blind

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The painting on the right was done by a child who became able to see thanks to cataract surgery after having been blind from birth. The leftmost panel is what he saw right after surgery; the middle panel 6 months later. (Luis Lesmes, Michael Dorr, Peter Bex, Amy Kalia, Pawan Sinha)

The painting on the right was done by a child who became able to see thanks to cataract surgery after having been blind from birth. The leftmost panel is what the child could see right after surgery; the middle panel 6 months later. (Luis Lesmes, Michael Dorr, Peter Bex, Amy Kalia, Pawan Sinha)

For the past few years, I’ve been dazzled by high-tech treatments for blindness. The first DNA Science blog post was about stem cells to treat  Stargardt’s macular dystrophy, and the most recent about gene therapy for  choroideremia, with the tales of several children becoming able to see for the first time in between. My book is about gene therapy for Leber congenital amaurosis. All rare diseases.

But I hadn’t heard of the astonishing Project Prakash until I read a short article, “Restoring Vision through ‘Project Prakash’: The Opportunities for Merging Science and Service,” in the December 17 PLOS Biology.

The brainchild of Pawan Sinha, Ph.D., professor of vision and computational neuroscience in the department of brain and cognitive sciences at MIT, Project Prakash is a humanitarian/scientific effort that is enabling children and young adults in India, congenitally blind from cataracts, to see. They’re among the 20 million people worldwide who are called “curably blind” or “needlessly blind.” Poverty prevents most of them from being able to see.

A congenital cataract is a severe clouding and hardening of the lens. (National Eye Institute)

A congenital cataract is a severe clouding and hardening of the lens. (National Eye Institute)

Dr. Sinha grew up in New Delhi, and went to graduate school at MIT. He started Project Prakash in 2002 after a trip to rural villages in India where he witnessed the sad scope of the problem of congenital cataracts – a very treatable condition. A grant from the National Eye Institute enabled him to assemble the team to provide cataract surgery, costing $300 per patient. He tells the story vividly in Scientific American.

“Prakash” is Sanskrit for “light.” So far the project has screened more than 40,000 children in India and treated more than 450. And it’s a small group. “The Prakash team includes 10 scientists, 5 clinicians, and 5 outreach personnel. It has been very gratifying to forge this collaboration across national boundaries and between clinicians and scientists,“ said Dr. Sinha.

A child born blind in India faces enormous stigma, and many parents do not pursue treatment – or don’t realize it’s even possible. In the U.S., a child born with cataracts would have surgery before his or her first birthday, leaving plenty of time for the nervous system to learn to integrate visual images into meaningful perceptions. Still, the surgery isn’t as easy as it is for the average older-age cataract patient in the U.S., who loses vision over many years and recovers it rapidly because the brain once learned to interpret images. The ophthalmologist breaks up and removes pieces of the hardened, opaque lens and replaces it with a synthetic lens. For children that means general anesthesia and a one-to-two day recovery. My mother was in and out for her cataract surgeries in a day.

Project Prakash is “a joint scientific and humanitarian effort.“ The humanitarian part is obvious. The scientific gain is in following a unique pediatric cohort who can reveal how a visually naïve brain begins to process and integrate images. Such research is typically done in infants, who can’t communicate what they’re seeing in a way that an 8 or 12 year old can. The oldest patients are in their twenties.

The participants in Project Prakash are challenging a long-held idea: that age of 7 or 8 is the upper limit beyond which a brain attached to sightless eyes can no longer become able to see. But the project has shown clearly that even a person who hasn’t seen for 15 to 20 years, since birth, can begin to make more visual sense of the world.

Visual_cortexThe investigations include behavioral observations, such as visual acuity and facial recognition, as well as non-invasive brain imaging to follow responses of the cortex to new visual information. The researchers used iPads to test contrast sensitivity.

Becoming able to see after removal of congenital cataracts is profound, but not instantaneous. At first, a newly-sighted person sees vague parts of a scene that, just shapes, that over time come into focus. By six months, shapes corresponding to different colors begin to emerge. “Our results show remarkable plasticity and vision continues to improve in many children long after the surgery,” said Peter J. Bex, PhD, a member of the Prakash team from the Schepens Eye Research Institute.

One particular type of experiment hit home with me, because I have synesthesia. This is a mixing of the senses that is especially common among writers and other creative types – to me, days of the week have specific colors and textures. My brain stamps a visual perception on a time concept. Synesthesia is thought to reflect an unusual overlapping of the cortical areas devoted to multiple senses.

Dr. Sinha and colleagues discovered that two days after children have cataract surgery and became able to see, they really can’t, in the sense of recognizing objects. A child could, with vision blocked, touch two objects and describe and discriminate them. The eyes couldn’t recognize an object that was quite familiar by touch. But it took only a week for those senses to merge. That wasn’t expected to be possible. An article just published in
PNAS Early Edition describes some of the recent results.

It’s wonderful that there is so much going on in vision research that I write about it often but can’t keep up. Check out the Foundation Fighting Blindness website. Stem cell therapies. Gene therapies and the spin-off of “optogenetics” that endows various cell types with the ability to sense light. The bionic retina. New drugs for age-related macular degeneration. And of course Project Prakash.

I think my favorite pair of words is “formerly blind.” I’m glad we’re hearing it more often.

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Gene Therapy News: Brain, Skin, Eye

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(Jonathan Bailey, NHGRI)

Several recent reports on ongoing clinical trials for gene therapies indicate that even preliminary studies with only a handful of patients can yield results with the potential to alter the course of the entire field. So after each description below, I offer a DNA Science “lesson learned” assessment: why the study is important.

INTO THE BRAIN – PARKINSON’S DISEASE

Gene therapy typically delivers a functioning version of a gene to cells needing it. Investigators Stéphane Palfi MD of AP-HP, Groupe Henri-Mondor Albert-Chenevier in Créteil, France and Roger Barker, PhD, at Addenbrooke’s Hospital in Cambridge, UK, have expanded the reach of gene therapy by delivering the trio of genes whose encoded proteins enable cells to make dopamine, the neurotransmitter that’s depleted as Parkinson’s disease (PD) progresses. Preliminary results on the gene therapy appear in The Lancet. Oxford Biomedica, a company developing “gene-based medicines,” is funding the trial of the triplo-gene therapy for Parkinson’s, called ProSavin.

Gene therapy enables cells of the striatum to use the 3 genes that make dopamine.

Gene therapy enables cells of the striatum to use the 3 genes that make dopamine.

In a healthy brain, neurons in the substantia nigra make dopamine. Their axons project to the striatum, where they release the neurotransmitter so neurons there can sop it up. Three enzymes control dopamine synthesis: two convert the amino acid tyrosine to levodopa, and a third converts the levodopa to dopamine.

Treating PD is an ever-changing question of balance. Oral levodopa can offset the dopamine deficit, but after a few years, motor symptoms develop. These include uncontrollable movements (tardive dyskinesia) and “on-off phenomena,” which are periods of improved mobility interspersed with periods of impairment, sometimes severe.

Where there are missing enzymes, gene therapy
is an option, and several have been tried for Parkinson’s disease. The safest gene therapy vector (a disabled virus that delivers the gene), adeno-associate virus (AAV), can’t carry a very large payload, only one smallish gene at a time. So the researchers turned to a larger vehicle to deliver the trio of genes, the lentivirus that causes swamp fever in horses, equine infectious anemia (EIA) virus. Many gene therapy experiments use a more familiar lentivirus – HIV.

A horse virus delivers Parkinson's gene therapy.

A horse virus delivers Parkinson’s gene therapy.

Instead of delivering the genes to the substantia nigra neurons that normally make dopamine, the gene therapy infuses gene-loaded viral vectors into both sides of the brain, right into the striatum. The resident neurons then, presumably, pump out what’s needed, even though they don’t normally do so. The goal: to “convert striatal cells into so-called ‘dopamine factories,” the researchers write.

The horse virus seems to offer the optimal combination of features. It doesn’t have the image problem of HIV, nor does it insert into oncogenes, causing cancer, as other retroviruses can do and have done in gene therapy trials. EIA also targets neurons, which don’t divide.

In the trial, 15 patients with advanced PD received low, medium, or high doses, while continuing to take levodopa. And so far it’s all good, up to 4 years later. ProSavin appears to be safe, and all of the patients reported improvements by 6 months. Eleven of the 15 needed to decrease the levodopa, with those in the highest gene therapy dose group needing the most reduction – suggesting that the little dopamine factories work. But the researchers caution that the results of the uncontrolled trial could be due to a placebo effect, something that’s been seen before in Parkinson’s research.

Lesson learned: Gene therapy can deliver components of a pathway – not just a single gene.

Stem cells nestle in the bulge regions of hair follicles.

Stem cells nestle in the bulge regions of hair follicles.

WHEN SKIN COMES UNDONE – EPIDERMOLYSIS BULLOSA
The skin is much more than a surface to smear make-up on. In addition to holding our insides in, it regulates body temperature, lets wastes out, keeps water in, and activates vitamin D. A square inch of skin houses 650 sweat glands, 20 blood vessels, 1000 or so nerve endings, 60,000 pigment cells, and a bunch of hair follicles.

About two-thirds of the way down a hair follicle lies a region called the bulge that houses stem cells that have the ability to divide to give rise to either hair or skin. These stem cells were discovered when physicians who treat severe burns noted that new skin forms around hair follicles.

In a group of inherited disorders called epidermolysis bullosa (EB), the layers of the skin separate. The skin is very fragile and blisters easily. Mutations in any of several genes cause EB, and subtypes are classified by the extent to which skin layers pull away from the basement membrane that normally separates the epidermis from the dermis beneath.

EB blisters the skin.

EB blisters the skin.

About 70% of affected individuals have the “simplex” form of EB that usually peels skin from the hands and feet. It’s manageable, and often several family members have it. Another 25% have the dystrophic form, with more widespread blistering that is replaced with scars that gradually tighten the body. Only about 5% of people with EB have the junctional form, in which the coming apart of skin layers is everywhere, even inside the throat. It can be deadly. Treatment for EB relieves symptoms, and bone marrow transplants have helped some children with the dystrophic form.

Seven years ago, Michele De Luca, MD and his colleagues at the Center for Regenerative Medicine at the University of Modena and Reggio Emilia, in Italy, sampled stem cells from the palm epidermis of a 37-year-old man named Claudio who has junctional EB. They used retroviruses to give the stem cells working copies of the gene encoding laminin 332-Β3, a linchpin-like protein that fastens skin layers. The doctored cells were grafted to the man’s thighs.

A year later, the grafted areas on the man’s legs looked pretty good – no blisters, infection, itching or inflammation, plus normal color and sensations. The healed skin had normal laminin adhering the layers, while surrounding skin was still ulcerated. Three years later, when the man hurt himself, his cut leg skin healed as if it had always been there.

The researchers waited 6 ½ years, to allow the grafted stem cells to go through about 80 division cycles, to see what would happen. The areas still look terrific, but the analysis, published December 26 in Stem Cell Reports, held a surprise.

It wasn’t terribly surprising that it took only a few gene-boosted epidermal stem cells to heal the legs – just 5 to 10 stem cells per 10 square millimeters, about the size of a large pea. The resident keratinocytes made the laminin, indicating that the stem cells had done what stem cells do: divide, differentiate, and replace, while maintaining the small population of stem cells to keep things going. (Many media reports that define stem cells as “turning into any cell type” ignore the more important function of self-renewal. If a stem cell doesn’t self-renew, it isn’t a stem cell.)

The grafted stem cells retained molecular memory of their origins in the palm.

The grafted stem cells retained molecular memory of their origins in the palm.

The surprise was that the stem cells taking up residence in the man’s legs bore a biological memory of where they’d come from – the palms. Not only was the new skin thick like palm skin, but it produced keratin 9, found normally only in keratinocytes in the soles and palms. “This finding suggests that adult stem cells primarily regenerate the tissue in which they normally reside, with little plasticity to regenerate other tissues,” De Luca said.

Lesson Learned: Stem cells aren’t a blank slate; if they are moved, they can retain echoes of their origins.

Corey Haas, who can thank gene therapy for his vision. (Foundation Fighting Blindness)

Corey Haas thanks gene therapy for his vision. (Foundation Fighting Blindness)

CORRECTING ANOTHER EYE DISEASE — CHOROIDEREMIA
Gene therapy has been making headlines in ophthalmology since 2007, when the first young people began to see the world for the first time after receiving working RPE65 genes to treat Leber congenital amaurosis type 2 (LCA2). Nearly 300 people have had that gene therapy, in several clinical trials. Check out this DNA Science post from November: Another Blind Boy Sees the Light Thanks to Gene Therapy.

Last week Robert MacLaren, MD, PhD, professor of ophthalmology at the Nuffield Laboratory of Ophthalmology, University of Oxford and colleagues published early results that gene therapy works for a different form of inherited blindness, choroideremia. That report is also in The Lancet.

The mutation behind choroideremia is in a gene called CHM, which is on the X chromosome. In the 1 in 50,000 people who have the condition, degeneration extends through several layers of the retina, in a patchy pattern. Matt During, MD, PhD, a professor of neuroscience at Ohio State University Medical Center and designer of the viral vector used in the clinical trial, described choroideremia when he told me about the exciting results last week. “A teenage boy will start losing night vision. Later he loses the peripheral visual field and then central vision, until he’s legally blind in his 50s.” I wrote about the technical details at Medscape Medical News.

The small gene, isolated affected body part, and gradual clinical course make choroideremia a perfect candidate for gene therapy. And the astounding success of the LCA2 trials indicated that even patients with just an “island” of photoreceptors left can improve.

Discovery of Gavin Stevens' LCA gene is the first step towards gene therapy. (Jennifer Stevens)

Gavin Groupies is funding research into developing gene therapy for his form of Leber congenital amaurosis. (Jennifer Stevens)

Like the Parkinson’s trial, the blindness trial was open label with escalating doses, delivering the gene aboard the small-capacity adeno-associated virus 2 (AAV2). Billions of vectors were slipped beneath the most sensitive part of the retina in 6 men, their untreated eyes serving as controls. But their retinas had to be locally detached to deliver the genes. One reason for the phase 1/2 clinical trial was to assess recovery from the detachment. Not only did the retinas quickly slip back into place, but the men reported improved visual acuity and light sensitivity in the treated eyes.

The results were better than expected. “When we started, our hypothesis was not to get recovery, but just to arrest progression, and it might take one to two years to see that. We weren’t expecting such early and dramatic improved function,” Dr. During said. That’s why they published so soon.

If the promising results persist, the gene therapy will be done on younger patients, who would likely do even better because they have more “islands” of preserved photoreceptors than older patients. And in the future, genetic testing could identify boys who will be affected and perhaps gene therapy deployed to prevent visual loss.

(For updates on gene therapy clinical trials for eyes, see the terrific tables from Irv Arons, a former consultant to the ophthalmic industry. They include Leber hereditary optic neuropathy, wet age-related macular degeneration, Stargardt’s macular dystrophy, achromatopsia, and forms of retinitis pigmentosa.)

Lesson Learned: The retina can be detached to deliver gene therapy, and recover. Fast.

I’ll describe other recent gene therapy successes in the March issue of print Scientific American. And maybe we’ll hear from next week’s Phacilitate Cell and Gene Therapy Forum in Washington, DC. Also check out my gene therapy book, which tells the story of one of the first patients to become able to see thanks to gene therapy – 8-year-old Corey, now poster boy for the Foundation Fighting Blindness.

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SyFy’s Helix: Tired Plot, Bad Science, Fun

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128px-Enveloped_helical_virus.svgLast week  I trashed Dan Brown’s Inferno for its poor use of science in the plot. But Inferno earns an A- in originality compared to the SyFy TV series Helix that debuted last Friday night, January 10. It’s another escaped-virus situation, but with a less creative setting than Inferno’s eerie indoor lagoon.

However I liked Helix, mostly because the first episode ended with the best shower scene since Norman Bates offed Janet Leigh in Psycho.

My gripe isn’t that popular fiction and TV base plots on science, that’s great. But why can’t they take the trouble to get details right? The plots of medical thrillers like Inferno and science fiction like Helix can fail when writers change or oversimplify scientific facts. This inevitably leads to breaking Isaac Asimov’s rule: change one thing.

THE PLOT
As Helix opens, a contagion is spreading in the lab facilities of mysterious underground biotech company Arctic Biosystems. A minor character refers to it as “big pharma,” but a lab under ice with a few dozen people running around isn’t like a huge corporate campus in New Jersey.

Is the mystery virus Ebola?

Is the mystery virus Ebola?

Like in Inferno, the protagonists in Helix know right away that they’re dealing with a viral pathogen, but are confused over the identity. At first they call it a retrovirus, and how this is determined from a bunch of dead bodies and an oozing live one isn’t clear. Maybe it just sounds cool. But soon a character states, “We have no idea what this thing is.”

Names are dropped. Ebola. Rabies. Marburg. Then someone suggests it is an Elisa virus, which I chalked up to being an intentional nerd joke. The reference goes by quickly, so perhaps the biologists in the audience won’t think of ELISA, the common technique to detect molecules that elicit an immune response.

Whatever the virus’s identity, it apparently causes rodents to develop without sex organs, and then to frantically hump one another. This developmental anomaly, we are told, is due to a defect in signal transduction. I don’t recall ever reading about that pathway.

Anyway, the facility is in the part of the Arctic that is international so the FDA can’t institute those pesky regulations. My husband Larry, a PhD chemist who does some mining work and knows about things like geography and geology, pointed out that such a lab, given the map shown, would either be land and part of Canada, Russia, or Greenland, or ice and prone to sinking when things warm up a bit.

How would you build it? There are no roads, no ships. How would they get all that shit up there?” Larry wondered. I’m sure we’ll find out in future episodes.

The researchers at the station have access to various areas using implanted identity chips, like my cats have.

Part of the plot is a love triangle. The head of the CDC’s Special Pathogens Branch, Dr. Alan Farragut, races to the mysterious station to check out his infected brother Peter, who “works in mutagens,” according to the show’s website, which I imagine must be very dangerous indeed.

More importantly, the infected brother beneath the ice floe bedded Alan’s wife, Julia (Jules) Walker, in the recent past. Dr. Walker is a senior scientist and co-head of the CDC’s Rapid Response Team. Barbs and bickers lingering from the affair annoyingly intrude on the plot amid annoyingly loud music.

Adding a dose of tension is the nubile young Dr. Sarah Jordon, about whom the website states, “What she lacks in experience she makes up for in audacity and medical knowledge.” More on her in a moment, but her advanced degrees are not as important as the fact that she will surely turn the romantic triangle into a quadrangle.

Love triangles get in the way in good sci-fi, although I suppose they might expand the audience. If Dana Scully and Fox Mulder had made out, for example, the X-Files  would have suffered an early death. (And I’m an expert here. I had a letter published in the end-of-the-year issue of the journal CBS Soaps pointing out that the ongoing plot on The Young and the Restless about one character dying to provide corneas for another because they are a tissue match doesn’t make sense because corneas don’t need to match.)

The virus-ridden Peter inexplicably becomes very strong and starts traipsing around the facility, rocketing up air vents like Spiderman. That’s dangerous. “Peter may have antibodies! We gotta find him. No one is safe from the virus until we contain him,” laments Alan. But Peter has been infected for under 48 hours, and it takes at least 5 days to make antibodies. Anyone remember when early HIV tests detected antibodies three months after infection?

Of course, there’s a bad guy. He looks like the artists’ depictions of human bodies in the human anatomy and physiology textbooks I write – people who represent every possible ethnic group. His name is Hiroshi Hatake, head of the Arctic Biosystems frigid facility. Near the end of the first episode, he removes his contact lenses to reveal alien eyes, reminiscent of the scene in V in which the supposedly human woman peels off her face to reveal the shimmering green reptilian integument beneath. Nice touch.

I’ll mention a few specific things I found disturbing with the pilot episode.

Death by Dionne?

Death by Dionne?

DIONNE WARWICK AND THE BROAD STREET PUMP

The episode opens with a scene of devastation in a small laboratory, with a few dead people and, most alarmingly, an iPod playing “Do You Know the Way to San Jose?” One guy isn’t quite dead – that’s Peter. Rivulets of black fluid ooze from his mouth, meandering down his neck. This seemed familiar, so I googled it and discovered the insidious black goo from last year’s Prometheus. That black goo was an agent of instant genetic change.

As the black ick drips from the sides of the victim’s mouth, we see something reminiscent of another Ridley Scott film, Alien, bulging from Peter’s neck. Or is it a moving goiter?

The famous Broad Street Pump, which any epidemiologist would recognize.

The famous Broad Street Pump, which any epidemiologist would recognize.

The next scene flashes to Alan at the CDC lecturing to a group of newbies. He’s dramatically telling the Broad Street Pump story, of how Dr. John Snow traced the 1854 cholera epidemic in London to a water pump, founding the field of epidemiology. It’s a classic tale, yet the audience of new Epidemic Intelligence Officers, who are mostly MDs, gasp in astonishment as their fearless leader holds up a piece of the pump. Music soars.

Reality check: the folks in the Epidemic Intelligence Service know the Broad Street Pump story. Consider eligibility requirements. This is too transparent a device to educate viewers – preaching to a group of tourists would have made sense. (On the subject of cholera, one of my favorite books is The Ghost Map by Steven Johnson and the CDC has a short account of the London outbreak.)

(Dept. of Energy)

(Dept. of Energy)

THE BIOGENETICIST

Last week I lamented the poorly-defined genetic engineers running around Italy in Inferno, wondering why they hadn’t gotten degrees in molecular biology or genetics like the rest of us. Helix is worse.

The Scene: young and precocious Dr. Sarah Jordon, clad in white jumpsuit and blue visor, has corralled two resident scientists, a nondescript 40-ish white male hematologist and a pretty light-skinned black woman with great hair who’s a biochemist. Kudos for logical specialists. But they’ve been exposed to the virus, so Sarah, age 26, is lecturing them on the danger, like they wouldn’t know. The biochemist says “What are you? 15?

And so Sarah reels off her list of accomplishments: 2 masters degrees and a PhD in biogenetics from MIT!

Biogenetics? What the heck is that? My degree is in genetics, no bio. Can one get a degree in abiogenetics where you study only DNA outside of organisms? But wait a minute — viruses aren’t organisms. Which leads to …

THE BINOCULAR ELECTRON MICROSCOPE

Next we see biogeneticist Sarah watching Jules, who’s peering through what looks like a binocular light microscope, the type you use in Bio 101.

Anything from the first set of cultures?” serious Sarah asks.

The cells are heavily damaged. I see filaments, cylinders, spheres, even icosahedrons!

Sarah makes a speech about ancient viruses from Greenland from 140,000 years ago. I googled this one – the virus can indeed be deadly, if you are a tomato. And 140,000 years ago doesn’t seem that ancient.

Look at that – right there .. it’s only 15 nanometers!” exclaims Jules, and Sarah runs over and they gaze enraptured at a computer screen that shows oscillating wormy things, shaped like helices. (Hence the show’s name – a helical virus, not a double helix, although the subtle purple of the “X” in the show’s logo suggests a future dual meaning.)

Here’s the problem. A 15-nanometer virus is considerably below the resolution of a light microscope, with which Jules is apparently working, yet anything placed in the vacuum of an electron microscope would not be gyrating. And the contraption doesn’t look anything like an electron microscope.

(CDC)

(CDC)

THE GOOD, THE BAD, AND THE DISGUSTING

Helix gets some things right. The outfits are quite nice, in appealing shades of teal and maroon, and the female characters look like Zumba instructors. Their make-up is perfect. The blue visors are attractive, but hardly barrier enough to keep out the black viral-ridden upchuck splash hurled from the infected.

In some scenes the female characters trade their Zumba outfits for clingy low-cut tank-tops. Is it important, when attending a viral outbreak beneath an iceberg, to expose cleavage? When Sarah donned such an outfit, her hand began to shake, reminding Larry (my husband, bravely watching Helix with me) instantly of the Gene Wilder character in Blazing Saddles.

The show has the typical illogic of characters venturing into dangerous situations alone, something even Law and Order: Special Victims Unit’s Olivia Bentson does. If I were in a lab under a glacier occupied by virally-infected zombies, I’d support the buddy system.

128px-Biohazard.svgIn one scene a veterinarian (an older, overweight blond woman destined for the Rosemary Clooney role from the Poseidon Adventure), who is alone in the scary animal facility full of escaped pissed-off oddly human-like monkeys (after all, a large sign says “transgenics”), encounters a missing infected crew member, who begins to babble in science-speak, so we can grasp some of what’s going on. “ .. activate replication cycle, add some genes! The perfect bioweapon. You can’t make a virus and expect it to follow instructions!” The zombie attacks the blond vet and throws up black on her, then rolls around moaning “What’s wrong with me?” So we’re on the road to another bioweapon story, like Inferno.

Elsewhere, some crew members inexplicably venture outside, where it is of course well below zero. No face coverings, gaping collars, for minutes on end, yet their visages don’t crack and slide off. The camera pans back over a landscape of frozen transgenic monkeys captured mid-scream, like the famous painting. I liked that. But I live near Albany, New York, where the below-zero temperatures that threw New York City into a tizzy last week can persist for weeks. We don’t stand around outside exposed.

The grossometer ranking for Helix is pretty high.

Larry’s favorite part, aside from the cleavage, was when Alan and Jules open two body bags to reveal skulls and black goo. These are Peter’s unfortunate colleagues. In response to the finding, Jules barfs into the biohazard suits that they’ve finally put on. As the chunks fly, splatter and ricochet, you can see that she had rather recently eaten.

Finally, we come to the shower scene, for which I noted foreshadowing. Earlier, to build the blooming tension between Alan and Sarah, she has a close encounter with the black-goo-dripping Peter, who does a Spiderman and vanishes through a ceiling vent. Alan grabs her shoulders in panic. “Did he get any secretions on you?” I nearly fell off the couch laughing.

So Jules is in the shower. But who is in there with her? Is it Bobby Ewing from Dallas, as in last week’s DNA Science post? Is it the disturbed Norman Bates from Psycho? Is it Dionne Warwick? No, it’s Peter, of the black spit. Weird music plays, a little like the theme from I Dream of Jeannie, to create surrealism. Peter moves in to kiss Jules, salivating ebony. And then in a smooch worthy of The Young and the Restless, the black goop comes pouring out of their adhered oral cavities.

As I await Jules’ vagina to seal and Peter’s peter to drop off, courtesy of the evil helical virus, I realize, with startling clarity, that Arctic Biosystems must have created the infertility-inducing virus from Inferno.

 

 

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Dan Brown’s “Inferno”: Good Plot, Bad Science

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The mad scientist is a dangerous stereotype -- an evil geneticist perhaps the worst.

The mad scientist is a dangerous stereotype — an evil geneticist perhaps the worst.

When Dan Brown’s latest novel, Inferno, was published last summer, several people insisted I read it – because it’s about an insane geneticist. So when my local library asked me to give a talk about a book with genetics in the plot, I chose Inferno. The talk is next week, and I had great fun marking up the book.

Dan Brown gets an A, as usual, for writing style. He keeps the reader turning the pages. But this time, the plot is a stretch, and he gets an F in genetics. (Warning: spoiler alert)

I learned that scientific accuracy shouldn’t get in the way of telling a good story at the Catalyst Workshop at the American Film Institute, where every summer a dozen scientists learn screenwriting from the pros. I went in 2005. After a week of dissecting The Day After Tomorrow, an exciting end-of-the-world thriller that seems all too possible with the recent crazy weather, we all concluded, as our instructors had said from the outset, that in entertainment, scientific accuracy just doesn’t matter. So it’s okay if the magnetic poles suddenly switch and a person recovers from septicemia in a few hours with one shot of penicillin. I love that film.

Tom_Hanks_as_Robert_LangdonAs anyone who’s read Dan Brown’s The Da Vinci Code and the Lost Symbol knows, protagonist Robert Langdon is a Harvard professor specializing in symbology who is summoned for emergencies that require him to rocket through Europe running from bad guys and heading off global disasters, while following clues and cues in art. He’s always called “Professor.” Many of my friends are professors, and I have an adjunct title myself, and we don’t call ourselves Professor. Maybe it’s different in the art world.

Having no time for anything other than math and science in college, I admit to being a dunce about the art part of the Dan Brown books. But I can comment on the science.

A MASH-UP OF 24, THE ANDROMEDA STRAIN, AND DALLAS

Robert Langdon (aka Tom Hanks) is reminiscent of Jack Bauer (aka Keifer Sutherland) in the TV series 24, in which incredible action unfolded over the course of a day. Inferno also takes place during a very busy day.

Due to the time pressure, Langdon’s metabolism is perpetually in high gear. Within a few pages, he was frozen in utter disbelief, startled, stunned, chilled, transfixed, he reeled, and did several double takes. I became concerned about his health when, in short order, his mouth fell open, heart raced, hair on his neck bristled, his pulse quickened, he sat speechless, audibly gasped, and barely breathed. Later on, his eyes went wide, as anyone’s would. His stomach knotted, he felt a visceral tremor, and his insides reverberated. Much of this happened whilst learning perfectly ordinary things about viruses, like the fact that they can nestle into our DNA.

Would my metabolism go into overdrive if I studied art? (Another of Brown’s very effective techniques is to put thoughts in italics.)

Poor RL. (I do like his initials. Me, Professor Langdon, and Ralph Lauren.)

Because the villain is a warped geneticist letting loose a human-gamete-eating virus, Inferno is also reminiscent of ContagionOutbreak, and The Andromeda Strain, the best of the genre. Actually The Hot Zone was best because it was true.

Illustration_for_Dante's_Inferno_by_Sandro_Botticelli_1481Inferno opens with a frighteningly vivid dream sequence based on artists’ depictions of Dante’s experiences in hell. Then RL awakens in a hospital room in Florence, tended to by a woman with a blond ponytail, Dr. Sienna Brooks, who gradually becomes just “Sienna” or Dantes_Inferno_Canto_28_“Ms. Brooks” while the other notables retain their titles. An obsession with feminine follicles emerges when we encounter a threatening “spike-haired woman” and the silver-haired head of the World Health Organization.

In addition to taking elements from 24 and the outbreak-type films, towards the end of the book, when the set-up is revealed, I was suddenly catapulted back to 1986 when Pam Ewing sees her supposedly dead husband Bobby in the shower on the TV show Dallas. Instead of introducing a surprise twin after actor Patrick Duffy quit and his character was offed and then he decided to return, the writers had Pam realize that her hubby’s death had been a dream, and the entire previous season, all 31 episodes, had never happened. (See Bobby Ewing in the Shower: An Epic Storytelling Gaffe).

Inferno has that feel of wasted reader time, especially when one is awaiting scientific details and explanations that never come.

THE PLOT

(Dept. of Energy)

(Dept. of Energy)

The plot point that sets everything into motion is when “genetic engineer” Bertram Zobrist explains the exploding human population problem to WHO’s silver-haired leader Dr. Elizabeth Sinskey. I assume Dr. Z has a PhD — he’s described as a “genius of genetics,” whatever that means.

(An aside: I hate the term “genetic engineering.” It’s a media invention. You can’t major in it, and it isn’t offered in engineering schools. The only place I could find it as an academic field is in a few online programs from outside the U.S., and in the name of a publication I used to write for. However, Dr. Brooks declares, “The world of genetic engineering is one I’ve inhabited … for many years… Perhaps she took the online course.)

Anyway, Dr. Sinskey doesn’t take Dr. Zobrist’s predictions seriously, perhaps because he is all gloom and doom with nary a mention of the “carrying capacity,” the leveling off of population expansion rather than annihilation of humanity.

So Dr. Z. hires a shady organization called the Consortium, led by the shady “provost,” which is headquartered on a big yacht full of mysterious bad guys and the spiky-haired woman. The Consortium is to shield him for a year as he invents and plants a viral plague to control population growth, without asking any questions.

Yersinia pestis, the cause of the Black Death.

Yersinia pestis, the cause of the Black Death.

Dr. Zobrist, it turns out, is a big Dante fan, and Dante died of bubonic plague. This is when I became intensely interested, having been criticized for writing an article for The Scientist on the plague genome, with a sidebar on using it as a bioweapon, just weeks after 9/11. (I also got in the mail at that time a letter from Libya that the FBI yanked as a possible anthrax missive.)

A plague story! Alas, Inferno evokes “plague” rather loosely. Somehow the plague bacterium that felled Dante along with a third of Europe during the Middle Ages morphs into an airborne viral infection in the novel. I’m not sure why.

Dr. Sinskey taps RL to find the bioweapon, because clues lurk in Sandro Botticelli’s depiction of Dante’s Inferno and other works. Snippets of the puzzle struggle to the surface of Langdon’s consciousness as he and Dr. Ponytail schlep through the art and artifacts of Florence. It would have helped the good Professor immensely, and saved at least 100 pages, if he owned a smartphone and didn’t have to bug tourists to use their Internet connections to look up the parts of Dante’s tome that he can’t recall.

Meanwhile, European CDC guys join the chase. Here is a good website for artistic clues and maps, if you’re like me and are just waiting for the science to show up.

Red_Herring (2) The plot detours to a few red herrings, which are jarring and manipulative. Dr. Brooks doesn’t really have a ponytail, it’s a wig; she’s bald from the lingering stress of having been raped. Dr. Sinskey isn’t really being drugged against her will when RL spies her slumped over in the back of a car; she has a barf disease that requires sedation. And finally, imposter Jonathan Ferris doesn’t have plague, he has a latex allergy from wearing a mask to play a character that appears earlier in the book. Also, good guys are bad guys and vice versa.

SENSELESS SCIENCE

Human_brain_female_side_viewEarly foreshadowing of superficial science is on page 36, where the author confuses cerebellum with cerebrum, and PET scans with CT scans. And he makes the classic trio of errors later on — human cell walls (animals are the only types of organisms without cell walls), “a bacteria,” and each of us having our own genetic codes (the correspondence between RNA codons and amino acids is universal, a mistake even in this week’s New Yorker article about genetics in China. We have individual genome sequences.)

But the worst illogic comes towards the end.

Tethered beneath the surface of a gloomy underground lagoon, not in Italy, lies a bag filled with yellowish-brown goop that holds enough of a mysterious virus to render much of humanity infertile – somehow. The investigators have set up PCR devices throughout the area, which all start blinking red to indicate detection of the “never-before-seen viral pathogen.” What did they use for primers? You can’t amplify a nucleic acid using the polymerase chain reaction without having a smidgeon of DNA or RNA from known pathogens.

Another error is one of omission. Sprinkled throughout the book are mentions of germline manipulation; Dr. Z. is a “germline genetic engineer.” We learn that fooling around with a germline is evil and powerful, but not exactly what it is. And herein lies the confusion. To a biologist, “germ” means “germ cell,” as in precursors to sperm and eggs. To a normal person, however, “germ” means a pathogen, such as a bacterium or virus. So it isn’t clear that a germline manipulation actually means changing future generations. And germline manipulation won’t even be necessary, because if the viruses work, there won’t be another generation. They need only obliterate existing sperm and eggs.

Sperm-20051108Practically, I had difficulty envisioning how the airborne viruses would get into gametes of the hundreds of people pushing to escape the lagoon, like Christmas shoppers barging into Wal-Mart. Germline manipulation isn’t easy. It’s banned in people for ethical reasons. In fact a gene therapy trial, for hemophilia, was temporarily suspended when altered genes turned up in participants’ semen, but luckily they were in the seminal fluid, not the actual sperm cells. Germline manipulation is used, however, to create animal models of human disease.

Fortunately, Dr Z’s scourge won’t drive humanity to extinction, because a few people are randomly immune to the virus. Random? How does that work? Wouldn’t a resistance genotype be quite specific, invented by those engineers, and not random?

Most vexing was the unfamiliarity with the ways of viruses. For example, the supposed experts in Inferno use the term “vector” as if it is a type of virus, like a herpesvirus.

128px-Biohazard.svg“Vector” is a general term that means a vehicle to transfer DNA. Proclaims Dr. Brooks, “A vector virus … rather than killing its host cell … inserts a piece of predetermined DNA into that cell, essentially modifying the cell’s genome.” I don’t know what predetermined means (viruses can’t think), and a virus inserting into a chromosome, a phenomenon called lysogeny, doesn’t modify a DNA sequence, it adds to it. And it’s normal. Our chromosomes carry loads of viral sequences.

Still, “Langdon struggled to grasp her meaning. This virus changes our DNA?

Alas Drs. Brooks and Sinskey can’t fathom how to counter the virus, other than making another virus to fight it. Antisense? RNAi? Biotechnologies to silence genes have been around for decades. One paragraph describes gene therapy, not calling it that, and claiming it’s new. It isn’t. The first clinical trial was in 1990.

The_X-Files_title_logoThe lack of savvy about viruses may be due to a Dana Scully effect, the assumption that any medical doctor is also a scientist. (She was from the X-Files, an MD constantly calling herself a scientist.) The three technical experts Dan Brown thanks in the preface are MDs – two are infectious disease specialists, the third I couldn’t find, and none belong to the American Society of Human Genetics. He needed to run the genetics parts by a geneticist. I would have happily done it.

Evolution is also handled oddly, although RL claims to be skilled in matters Darwinian. He and the docs confuse natural selection and survival of the fittest, which deal with reproductive success, with genetic enhancement to “advance the species” and “create better humans.” I think Brown means that those who survive to have successful sex after the viral scourge will then, either by the viral DNA or some new genetic treatment, churn out kids who get high SAT scores and humanity will be saved. The long-awaited explanation of the science is delivered in a style I call hand-waving — throw out a bunch of terms that presumably readers won’t recognize to make it sound like it makes sense. It doesn’t.

Even the conclusions in plain English are vague to the point of meaninglessness: “He had unlocked the evolutionary process and given humankind the ability to redefine our species in broad, sweeping strokes.” From a virus with a predilection for gametes?

Finally, the fake-ponytailed Dr. Brooks, flummoxed by the ins and outs of viruses, assures us that “the human genome is an extremely delicate structure … a house of cards.” Then how can engineers manipulate it with a whiff of a virus?

It’s great that a novelist as acclaimed as Dan Brown would base a plotline around genetics. But he squandered an opportunity to teach the public about the good that geneticists do. Why not a subplot of a sick kid? A family that uses the terrifying “germline genetic engineering” to vanquish a terrible genetic disease?

The last thing our science-phobic world needs is another mad scientist – even a fictional one.

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