ALS Treatment (in Cells) – Too Late for Glenn, But Wonderful News

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Glenn Nichols and the hospice team.

Glenn Nichols and the hospice team.

I was cutting and pasting a post about Sunday night’s episode of Call the Midwife, which was about cystic fibrosis, when a news release came in that brought me to tears.

Kevin Eggan and co-workers at the Harvard Stem Cell Institute have discovered that the seizure drug Potiga (retigabine), FDA-approved in 2010, apparently tempers the hyperexcitability of neurons derived from induced pluripotent stem (iPS) cells made from patients who have amyotrophic lateral sclerosis (ALS).

Of course clinical trials are necessary to test the drug on patients, not just their derived cells. And because this blog investigates perspectives beyond the headlines, I’ll leave it to others to explain the exciting science. Instead, this post is a tribute to all researchers who work on neurological diseases, from Glenn Nichols. He died of ALS several years ago.

Glenn was my favorite hospice patient. I was paired with him as a volunteer because he was an English teacher who wanted to write his memoir, before ALS took away his speech.

When I met Glenn, he wasn’t expected to live more than 6 months, but he survived for 14. I like to think that his writing kept him going. During that time, I’d type away as Glenn’s life story poured out. We grew close, and at times I feared he was going to ask me to help him end things. He never did.

800px-Allman_Brothers_Band_-_Gregg_AllmanSo we wrote his memoir. Then we spent three weeks discussing the end of life. He was ready. But then we both noticed that he was still very much alive, still able to talk, still able to eat – even peanut butter! So he asked me a favor – he’d always wanted to write a novel. And so we did. It was the quintessential midlife-male fantasy: Glenn was a member of the Allman Brothers, riding a motorcycle with his wife on the back, her long black hair flying behind her like a flag.

I learned after Glenn passed away that she absolutely hated the book. But at the funeral there was her photo, in her twenties, riding in a convertible with her dark mane behind her like the tail of a comet.

One of Glenn’s wishes was to be published. I knew the editor of our local newspaper, and so I edited some of Glenn’s memoir, and one Sunday, there it was on the front page. So I am typing it in here, so Glenn can live on. I know that the repurposed seizure drug is a very early-stage discovery, but after so many disappointments in treating this terrible disease, now there is hope.

Schenectady Gazette, September 16, 2007

Lou Gehrig’s Disease Saps The Body, But Person Inside Is Still There
By Glenn Nichols

On October 25, 2005, my life as I knew it came to an end when the doctor said the words “amyotrophic lateral sclerosis.”

I didn’t hear much after the prognosis of three to five years from diagnosis, as I tried desperately to remember when I started having problems. How much time did I have?

The news wasn’t a complete shock. I’d searched the Web, gone from doctor to doctor, had test after test. ALS, or Lou Gehrig’s disease, kept coming up.

256px-Motor_Neuron_Before_Post-Polio_SyndromeALS is a fatigue-driven terminal illness. The neurons in the voluntary muscles continually fire, until the muscle is destroyed. For some people it starts in the throat, with excess saliva and then trouble swallowing. I have the other type. It began with tingling in my right hand and forearm. That led to carpal tunnel surgery, for a diagnosis of ALS is one of exclusion and usually a last resort.

My hands continued to worsen, the fingers curling as my muscles shrank. I had fasciculations – muscles twitch, and you can actually see it and feel it, like snakes slithering, painlessly, beneath the skin.

My strength sapped away. I couldn’t button shirts or zip zippers. Weakness became a major problem, because as a writing teacher, I could no longer manipulate the markers on the whiteboard. I had to have a student do it.

Then strange things started to happen in my lower parts. I ran as if I had clown shoes on, and my feet flapped. My back was growing stiff, and my spine curving. Visiting my primary care doctor and then a neurologist led to another misdiagnosis, a pinched nerve, but then a neurosurgeon saw what was wrong simply by watching me walk unclothed.

From the first tingling in my hands until accurate diagnosis was four years, by which time I’d lost all faith and trust in the medical community. Then I had the good fortune to be referred to the regional ALS center at St. Peter’s Hospital in Albany, and Dr. Jonathan Cooper. Soon a nurse from the center called to set up a meeting at our home. She brought a wealth of information, patiently answered all of our questions, and when she left that day, my wife and I felt much better, knowing a team of experts would help us through what was ahead.

Three months later, I was walking with a cane; by early spring, crutches; by May, a manual wheelchair. My decline has continued in fits and starts, with periods of new difficulties interspersed with plateaus as I adjust to new limitations. Community Hospice of Schenectady came on board to help last January, providing daily visits from a nurse, aide, chaplain, social worker or volunteer.

Currently I am immobile and in bed, with a BiPAP machine to force air into my lungs. My muscles are dying, curling my hands and feet into useless claws. When I’m lifted from my bed, my back is so bowed I look down at the floor. But I can still eat and talk and even blog.

And I’m still me.

Only a small percentage of ALS cases are inherited. The first gene discovered was that for superoxide dismutase (SOD1).

Only a small percentage of ALS cases are inherited. The first gene discovered was that for superoxide dismutase (SOD1).

A person receiving a terminal diagnosis is not the only one affected. It took me awhile to understand this. I knew how it would affect close family members. What I wasn’t prepared for was the reactions of others.

Bad news travels quickly, and out of the woodwork, people began to appear. Most didn’t know what to say. People I hadn’t seen in a long time would show up, but not bring up my health until I did. I’d have to tell them it was OK to talk about it, that it was a reality that I was living with. Others went in the other direction. Thinking they were doing the right thing, they’d festoon me with books about famous folk with ALS, such as “Tuesdays With Morrie” or books on death and dying.

That gets old real quick. So I told my visitors that even though I have to live knowing what’s coming, I’m still the same person. We can talk about other things: about music, baseball, horse racing, cars, idiotic TV shows.

Now that I’m bedridden and hooked up 24/7 to my BiPAP machine, I do look different. But somehow visitors equate this with being different. I remember the moment when a friend suddenly realized I was no longer able to walk. I could see it in his body language and in the look on his face. I told him that a lot had changed, but fortunately it doesn’t affect your brain or your personality. So just treat me like me.

Visiting a friend with ALS may be tough, but at least I’m not in pain. It’s different if someone has stage IV cancer, or dementia and they don’t recognize you. But visit. Talk about anything and everything. And remember that no matter what the person looks like on the outside, he or she is still your dear friend on the inside.

Be there.

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Signal Transduction: Poetry in Motion

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A signaling schematic (but not human due to cell wall).

A signaling schematic (but not human due to cell wall).

When I was in school, the scary parts of biology were cellular respiration and the synthesis and degradation pathways of the 20 amino acids. Each of us probably has our own personal bionightmares. For today’s students it could be all those interconnected pathways that depict the signals, receptors, second messengers and beyond that enable cells to function and specialize.

STD to us doesn’t mean what was once called a venereal disease. It means signal transduction.

Given the staggering molecular details that underlie signaling, biology professor Robert Blystone of Trinity University in San Antonio was stunned when senior Kristen Gill, a biology major with an English minor headed to medical school next year, offered an astonishingly elegant and astute answer to a question. In their words:

120px-Cyclic-adenosine-monophosphate-3D-spacefillDr. Blystone: I was leading a class through a signal transduction exercise. I put in front of them the Wikipedia figure. I asked the students to prepare a not more than 100 word summary of the essence of the figure. Below is a student’s effort at the exercise.

Endless arrows
Endless molecules
Endless receptors

How can so much fit into one tiny cell?

How can so little create an entire organism?

External environment

External cues
Through the phospholipid bilayer

Internal cascades
Internal inhibitions
Internal inhibitions of inhibitors
To grant molecules access to the nucleus

Create the proteins that give rise to the
External environment
Internal cascades

1 cell, 2 cells, 4 cells, more
Divide, communicate, specialize, relocate
Repeat, repeat, repeat

120px-Cyclic-adenosine-monophosphate-3D-balls-2Brilliant, Kristen and thanks Dr. B. You’ve started something! I invite readers to submit creative DNA writing – haiku, sonnets, I once met a girl from Nantucket, anything goes.

A few posts coming up will highlight essays about DNA science from young participants in contests that I’m involved with. Teens’ comfort with DNA science is amazing. Much to my surprise, it turned out that the target audience for my gene therapy book was science-savvy 15-year-olds, according to a review  in School Library Journal. The Katniss/Tris crowd. My agent and I were astonished. But that explained the glazed eyeballs of audiences for my book talk who went to school before DNA’s discovery as the genetic material.



Today’s teens and twenty-somethings grew up familiar and comfortable with DNA science. I can’t wait to find out what they will accomplish within the next decade with all those genome sequences at their fingertips.

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A Challenge to the Supremacy of DNA as the Genetic Material

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85221_largeAbout a month ago, a news release stood out among the many I get every day: “A challenge to the genetic interpretation of biology,” from a physicist and chemist from Finland, Arto Annila and Keith Baverstock. They’d just published “Genes without prominence: a reappraisal of the foundations of biology,” in the Journal of the Royal Society Interface.

One sentence from the news release grabbed me: “The result is evolution from simpler to more complex and diverse organisms in both form and function, without the need to invoke genes.” Instead, Drs. Annila and Baverstock invoke thermodynamics.

I was mesmerized, mostly because I am immersed in writing the 11th edition of my human genetics textbook and a non-DNA-centric view got me thinking. So I read the paper and asked the authors to guest post. Their idea brought me back to pre-1953 thinking that proteins are the genetic material, mostly because we knew more about them than the mysterious goop on soiled bandages that was DNA.

Then last week I posted here about the information from a dozen sequenced human genomes not being all that clinically useful, at the same time that the blogosphere trumpeted the not-very-surprising finding that a gene attached to obesity was actually controlled by another gene. The news last week seemed to validate Drs. Annila and Baverstock’s concern about genome sequencing entering the clinic when we don’t fully understand how genes interact at the level of their products, the proteins.

Dr. Baverstock kindly agreed to post. His impressive bio is here. Most notably, he brought  to global attention the increased childhood thyroid cancer incidence in Belarus caused by radioactive iodine from the Chernobyl accident. (I had thyroid cancer although I’ve never been near a leaking reactor.) Here he shares his thoughts, lightly edited, subheads added:


Arto Annila and I are making the seemingly outrageous claim that mainstream biology, since around the 1920s, has pursued a course that is deeply flawed. Critical to that course is the notion that genes are Mendel’s units of inheritance and that their material realization is a DNA base sequence. We propose instead that Mendel’s unit of inheritance is a process involving the interaction of mainly activated proteins contributing to an attractor state that represents the phenotype. Many will find this language of physics unfamiliar. However, cells are complex dissipative systems (CDS) in that they consume energy and thus operate according to the 2nd law of thermodynamics as it applies to open systems.

A lily cell dividing. (Andrew S. Bajer)

A lily cell dividing. (Andrew S. Bajer)

First, two irrefutable facts in justification of our position:
1. When cells divide they inherit the state of the cell. If this were not the case, cancer and differentiation would have to be one-step processes. The state of the cell cannot be encoded on the DNA base sequence: it is the active proteome.
2. Key biological processes, such as development, growth and aging, are irreversible in time, whereas standard textbook physics describes time reversible deterministic dynamics.

It is very well known that at cell division the cytoplasm is partitioned between the two progeny, but not emphasized, as we propose, that it contains a coherent complex process of interacting proteins. When this state is understood as the unit of inheritance, the epigenetic memory that enables processes, like differentiation, to take place over several cell generations is a natural manifestation. In addition, CDS physics supports the phenomenon of quasi-stability – that is, stability within limits: attractors are quasi-stable states formed by the interacting proteins. This would mean that inheritance at the cellular level is not after all a matter for the nucleus, but rather for the cytoplasm.


The nucleus/cytoplasm issue was hotly debated around the turn of the century – not the last one but the one before, and eventually resolved in favor of the nucleus by the geneticist T H Morgan in 1926. It’s clear that components of the egg cytoplasm are inherited at fusion, the mitochondria for example, but it has generally been regarded that the sperm delivers only genomic DNA. However, studies on male fertility have revealed that proteins essential for successful fertilization are present in the sperm and some of the chromatin is in a non-condensed state and thus, possibly even active. Therefore, we can assume that the sperm is capable of supporting a protein-based attractor state.

Gibel_carpOne experimental way to resolve the nucleus/cytoplasm issue is cross species nuclear transfer to enucleated eggs. This has not proved possible with mammals, but has been successful with fish. Enucleated goldfish eggs transplanted with nuclei from carp eggs develop with the outward appearance of the donor carp, but with a vertebral number (26 to 31) consistent with goldfish (26 to 28) rather than the genomic DNA donor carp (33 to 36). We assume that when two dynamic attractors are placed in a common environment, as in the case of the zygote, that they will “synchronize” as, for example, with Huygens’ clocks. Therefore, we argue that biology can explain inheritance on the basis of a sound foundation in the appropriate physics, without resorting to mechanistic narratives involving genes.

Furthermore, work in the 1970s demonstrated that enucleated HPRT-competent (HPRT is an enzyme whose absence causes the awful Lesch-Nyhan syndrome, an inborn error of metabolism-RL) fibroblasts in vitro could correct HPRT deficiency in fibroblasts with an intact nucleus, by transferring molecules via gap junctions, without the need for protein synthesis. In addition, erythrocytes (red blood cells) dispose of their nuclei at the last stage of differentiation, but retain, for example, the circadian rhythm function for their lifetime.

In fact, the evidence clearly points to routine cellular function (apart from cell division) and regulation in somatic cells being a matter for proteins without the intervention of genes. If, for example, the dark/light rhythm changes (travel over a few time zones) then intervention involving new transcription to adjust the circadian rhythm does occur, but otherwise circadian rhythm is taken care of by protein chemistry, as has been demonstrated in vitro.


If you have read as far as this, you are no doubt wondering about the plethora of experimental evidence for the action of genes that has accrued since Mendel experimented with pea plants in the monastery garden in the mid 1800s. It is impressive, but how complete is it and what does it really explain?

The American geneticist Richard Lewontin drew attention in 1974, in a book on population genetics, to the fact that all experimental geneticists since Mendel had studied very marked, i.e., easily measured, traits, such as flower color. He identified the following paradox “what is measurable is not interesting and what is interesting is not measurable.” We suggest that these marked traits are rather special and they often do associate with gene sequences, but the association is not causal. A correlation or association as such does not reveal driving forces of ensuing effects. Key here is the thorny issue of protein folding.


Beta-meander1An important step in the Central Dogma (DNA encodes RNA encodes protein-RL) is the folding of the peptide to form the protein, which can become biologically activated and contribute, as a component of the attractor, to phenotype.

Anfinsen’s dogma, derived from experiments with the enzyme ribonuclease, says that the amino acid sequence of the peptide dictates the folding. Were that true the “protein folding problem” would have been understood by now. In fact, predicting the folded structure is still an unsolved problem and according to Arto Annila that is because the folding process is a dissipative (energy consuming) non-determinate process. It is non-determinate because of the involvement of the environment in which the folding takes place.

An extreme example is the involvement of chaperone proteins, which provide an environment favoring a specific folding. Therefore, we have the possibility that a single amino acid sequence, as a peptide, dictated by a gene coding sequence, can fold into more than one protein and therefore perform more than one biological activity: the determinate relationship between sequence and biological function, crucial to the Central Dogma, is violated. It is, of course, also violated by the several ways in which a single multi-exon gene sequence can be spliced to produce several peptides.

320px-Frozen_lake_(2152865126)EMERGENT PROPERTIES

Another aspect of the physics of dissipative systems is the role of symmetry breaking and the consequent emergence of new properties. Symmetry breaking may sound obscure, but it is a simple concept.

Liquid water has perfect symmetry in that no matter from which direction you look at the molecules, the view is the same. A perfect sphere has perfect symmetry for the same reason. If the water freezes to ice, the perfect symmetry is lost or broken and the property of rigidity emerges. In Finland, the lakes freeze over in the winter and roads across the lakes open up, exploiting this emergent property. In this case the symmetry is broken by a phase transition, but any transfer of energy has the potential to break symmetry and therefore to give rise to emergent properties.

We see this all the time in chemistry. If we take a mixture of the harmless and odorless gases, nitrogen and hydrogen, and heat them to a high temperature, exchange of electrons between the two molecules occurs (symmetry breaking) and ammonia is the product with the emergent properties of a noxious and pungent gas. If this reaction had never been performed, there would be no way to predict, from the physical properties of hydrogen and nitrogen, the properties of ammonia – its properties are emergent.


What we believe drives the cell to deliver its phenotype is protein chemistry – chemistry in which information derived from the folding process (not from the amino acid/DNA base sequence) is processed through the attractor to yield the very specific, but emergent, and therefore unpredictable even from knowledge of the proteins, let alone the DNA sequence, properties of the cell. So the sequence information in DNA serves only to specify the amino acid sequences of peptides; the emergent information that underpins the phenotype is not even primarily of the same type as the sequence information.

BRCA1Sequence information is usually regarded as being composed of “bits,” but the emergent information carried by proteins is physical in character. Consider a notice outside a café in say Tucson, Arizona. It says, in Finnish, that anyone is welcome to visit for a free lunch on Wednesdays. The proportion of Finnish speakers eating lunch in that café on a Wednesday is likely to be far higher than that in any other café in town. The information in the notice can of course be quantified in terms of “bits,” but that is irrelevant to the “physical nature” of the information that only Finnish speakers recognize. Enzymes express their activity by their ability to recognize a specific substrate with which they can react and we are suggesting that this kind of physical recognition process underlies the interactions between cellular proteins and thus, the operation of the attractor and therefore, cellular phenotype.

The attractor is also responsible for the regulation of the cell: that is why enucleated cells retain biological functions and even communicate and initiate functional activities, such as building gap junctions or exhibiting circadian rhythm. This forces us to the conclusion that causality in cells is exercised downwardly from the phenotype to the genotype (for example, to initiate transcription or even modify the genomic sequence), exactly the reverse of the Genotype to Phenotype (G -> P) concept underpinning population genetics.

However, if we think about the origin of life from a non-creationist perspective it is difficult to see how it could have been otherwise: the life process initiated itself and recruited nucleic acids in order to retain the necessary peptides as the cell’s raw materials. Recent evidence shows that in the period from 4.5 to 3.8 billion years ago, a great deal of carbon was delivered to the Earth via meteorites and that the shock of impact was sufficient to synthesize amino acids. Meteorites are also believed to have delivered bases. From the perspective of the physics of complex dissipative systems, it was almost inevitable, given the climatic conditions on Earth, that energy from the Sun, via the second law of thermodynamics, would concoct a form of chemistry we call life.

256px-1e7m_comparisonSo as astronomers discover ever-increasing numbers of planets, in and beyond our galaxy, orbiting suns in what is known as the Goldilocks zone, it seems inevitable that Earth is not alone in the Universe in supporting the phenomenon we call life. In the evolution of how we explain that phenomenon, genetics and genes have played a prominent, even dominant, role. Genetics is, however, only a statistical association between something we had to infer and something we could observe.

Medicine Vial with DNANow that genome sequencing is routine and we no longer have to infer the genotype, we can see things are not so simple. We are faced with either generating ever more complex genetics-based narrative explanations for biological behavior or looking for a more rational basis for biology: we opted for the latter.

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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.

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.)

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.

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.

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.

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.

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.



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.”

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.

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

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.

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.

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.


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.

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.)

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)

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.

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.

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.

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.

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.

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.

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.

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.

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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