A Checklist for Gene Therapy From the UK Cystic Fibrosis Trial

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A clinical trial is a series of hurdles.

A clinical trial is a series of hurdles, but not in parallel.

Washington, D.C.           I’m at the American Society of Gene and Cell Therapy annual meeting, one of my favorite conferences. The very first talk provided a great example of why it is taking gene therapy so long to reach the clinic — a milestone that hasn’t happened yet in the U.S. The first gene therapy experiment was 24 years ago.

For the first talk, Uta Griesenbach, PhD subbed last minute for Eric Alton, MD to present progress in a phase 2b double-blind placebo-controlled clinical trial of gene transfer (that’s what gene therapy is technically called before results indicate it works) to treat cystic fibrosis (CF). It’s “wave 1” of the effort from the UK Cystic Fibrosis Gene Therapy Consortium, in which more than 80 researchers are participating.

The consortium made headlines about two years ago when the UK Medical Research Council and the National Institute for Health Research refilled their coffers with £3.1 million (US$4.9 million). Funding had been dwindling, perhaps because of the physiological hurdles that CF presents, against the backdrop of gene therapy setbacks. These began with the death of 18-year-0ld Jesse Gelsinger in 1999 days after gene therapy, and the leukemia that’s cropped up in clinical trials for two immune deficiencies. Many investigators had given up on gene therapy for CF – but not the tenacious UK group, led by Dr. Alton.

Cystic fibrosis results from an absent or malformed chloride channel.

Cystic fibrosis results from an absent or malformed chloride channel.

Cystic fibrosis, at the risk of evoking clichés, has been a tough nut to crack. The gene and its encoded protein, the cystic fibrosis transmembrane regulator (CFTR), were discovered in 1989, and my post from April 10 details some of the earlier history of this disease.

The multi-system symptoms – lung congestion and susceptibility to infection, pancreatic insufficiency, male infertility – result from malformed, misfolded, or absent chloride channels. More than 25 clinical trials, involving more than 400 patients, have attempted to deliver functional CFTR genes.

Improvements were limited or transient, because of the nature of the illness. Thick sticky mucus gets in the way, and cells lining the respiratory tract divide so often that normal cell turnover may jettison an altered cell in a cough or swallow too soon to see a sustained effect. Plus, correcting the problem in the airways won’t alleviate the pancreatic clogging that leads to the classic symptom of “failure to thrive.”

Most gene therapy trials use retooled viruses as vectors to deliver the payload, but wave 1 delivered CFTR genes in an aerosol of tiny lipid bubbles, liposomes. They echo the lipid bilayer of a cell membrane so coalesce themselves across that barrier, releasing their cargo inside.

At first I was disappointed when Dr. Griesenbach said she wouldn’t be presenting results. But when she explained why – “we haven’t broken the blinding yet” – I suddenly realized that the journey to demonstrating that a new therapy works is just as interesting as arriving at that destination.

A double-blind, placebo controlled trial design may seem cruel, intentionally depriving people of a possible treatment, but it is essential to demonstrating that a new treatment actually works. There are workarounds – the control group can receive an existing treatment. And trials are revamped, access expanded or FDA approval accelerated if a result is obviously compelling and people are suffering. The cancer drug Gleevec is an example of a drug hurtling towards the market. It happens, but very rarely.

I don’t think Dr. Griesenbach intended to focus on the hurdles researchers must leap to even plan testing a gene therapy, but that’s what held my attention. The reasons help to explain why clinical trials can take years. Following are the questions that needed answers and the concerns that emerged during this CF trial, which has been in progress since 2002.

1. How much of a gene’s function must gene therapy restore?
In gene therapy, a small change can go a long way. That’s the case for a gene transfer approach for the clotting disorder haemophilia B, presented at a news conference by Andrew Davidoff, MD, from St. Jude Children’s Research Hospital. Introducing the gene for clotting factor IX that restores the level to less than 8% of normal activity can free a man from needing to take clotting factor to prevent life-threatening bleeds.

For CF, men whose only symptom is infertility have 10% residual function of the chloride channels. “So if we can achieve some increase, we can have a significant impact,” said Dr. Griesenbach. A 6% increase in lung function might be all that’s necessary.

A liposome is like a bubble of cell membrane.

A liposome is like a bubble of cell membrane.

2. How should researchers pick the best vector and its cargo?

Choosing a vector and making it safe is perhaps the toughest challenge in gene therapy. Investigators must design the delivery method before a phase 1 trial gets underway, and stick to it.

The situation isn’t like getting a new laptop when Apple introduces a new and improved model. Researchers can’t change or tweak a virus, alter the recipe for a liposome, or replace the DNA cargo without going back to square 1, phase 1. It’s one reason why the gamma retroviral vectors that caused leukemia and the adenoviruses that evoked a devastating immune response are still in use, although some have been made “self-inactivating.”

The CF trial used a liposome delivery method developed at Genzyme awhile ago. But the researchers modified the DNA within to decrease the stretches of cytosine and guanine (“CpG islands”) that invite inflammation and they added a bit to extend the effect. That meant starting from scratch in the phase 1 trial, even though the liposome recipe had been used before.

CF affects more than the respiratory system.

CF affects more than the respiratory system.

3. Which endpoints are the most meaningful?

The CF team tests cells lining the nose and airways for chloride transport, finding that it can reach about 20 percent of normal following gene transfer. Other assays include a “lung clearance index” from inhaling a harmless dust and scans that use technetium to show clear areas in the lungs.

But these measures meant little to the trial participants. “The patients said, ‘so what? Will it make my lung disease any better?” Dr. Griesenbach said. “Our program is now hinged around addressing that question. How much improvement is necessary to have a clinical effect?” A quality-of-life questionnaire is now part of the protocol.

4. Which types of patients should a clinical trial enroll?

Should the sickest patients try a new treatment because they are the most desperate, or should the healthiest, because they have a better chance of surviving the experiment? Part of the outcry over the death of Gelsinger that effectively halted the field for two years was the fact that he had not been desperately ill.

The symptoms and natural history of CF dictate the optimal age of trial participants. “In CF we face a dilemma. Very young children have less mucus, but it is harder to measure their increase in lung function. In the full-blown disease patients have lots of thick sputum. It is hard to find the right patients. You need a balance,” Dr. Griesenbach said. They decided on 12 as the minimum age, with average age 22.

Patients received 12 monthly doses, bracketed by 4 additional visits, and the last participant finished just two weeks ago. The monthly intervention was nothing compared to the hours of procedures that people with CF go through on a daily basis to expel mucus.

The CFTR gene is on chromosome 7.

The CFTR gene is on chromosome 7.

I don’t know whether the patients in the UK trial are stratified by mutation, but the development of the blockbuster drug Kalydeco illustrates the importance of distinguishing among the 1600 or so variations of the CFTR gene sequence. Kalydeco corrects misfolding, which affects only some patients with specific mutations, but can be teamed with other drugs to help more. And a new contender for a CF drug is targeted at patients with nonsense mutations, who make no CFTR protein at all.

5. Expect the unexpected.

The researchers determined that they needed 120 patients, and they started with 130, just to be safe. Then in January 2012, the FDA approved Kalydeco. Some participants, understandably, dropped out of the liposome gene transfer study to take the new drug.

6. Think ahead.

So far, CFTR delivery via liposomes seems to be safe. Some of the patients who are feeling better baked cakes for the researchers, although efficacy isn’t known yet. But the consortium is running a parallel “wave 2” using lentivirus (disabled HIV), in case the fatty bubbles aren’t efficient enough or the effect too transient. (I’ll cover HIV as a gene therapy vector in a future post.)

Results are in, Dr. Griesenbach concluded, and will be presented at the North American CF Conference in October. So far the team knows that patients experience a very brief period of fever and decrease in lung function, but recover well. Then some of them improve. A third of patients fully responded, another third had some correction of lung function but not to entirely normal levels, and a third didn’t respond.

The unblinding will reveal whether the gene transfer is responsible for the patients who did the best. And if they are indeed the ones who received functional CFTR genes, then the next chapter – a phase 3 trial – will be up to industry.

It’s easy to see why approval of a gene therapy takes so long!

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Of Tissue-Engineered Vaginas and Default Options

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A tissue-engineered vagina (Wake Forest Baptist Medical Center)

A tissue-engineered vagina (Wake Forest Baptist Medical Center)

I’m old enough to remember textbooks and biology classes that defined being female as a “default option” in human development.

If the ambiguous sex organ precursors in an embryo “failed” to follow the coveted male route, we became female.

If the SRY gene “failed” to turn on, we became she.

I’d always thought this male-centric teaching of human sexual development disturbingly close to Genesis: “The Lord God fashioned into a woman the rib which He had taken from the man. The man said, ‘This is now bone of my bones And flesh of my flesh; She shall be called Woman, because she was taken out of Man.’”

Lost in the discussion was the paradox of the puny Y chromosome and its meager roster of genes compared to the X. So I was happy when discovery of the Wnt gene pathway to femaleness finally came along and validated my existence, beginning the elucidation of the complex gene cascades that sculpt a female reproductive tract.

Yet the predominance of maleness continues.

To illustrate this post, I perused the offerings at Wikimedia Commons.

For vagina? Eight images, all tastefully anatomical.

For penis? 160! And that’s only the human ones. Wikimedia depicts male organs in various stages of alertness reminiscent of Moh’s scale of hardness in geology. The Wiki take on the male organ also offers artist’s renditions, eclectic enhancements, a handful of abnormalities, and a few action videos that I won’t go into.

(Wikimedia Commons)

(Wikimedia Commons)

With this sort of backstory, it’s little wonder that very young women who discover that their vaginas seem very narrow or short are confused, and then devastated when they learn they have a medical condition that affects the reproductive tract. Any variation on normal anatomy can be disturbing, but the situation is even worse when the affected body part is one we could barely even mention until Eve Ensler gave us the hilarious Vagina Monologues a few years ago.

If it was difficult to begin to talk about women having vaginas, it’s even harder to talk about women whose vaginas do not develop fully. But that’s the case for the 1 in 4,500 women with Mayer-Rokitansky-Kuster-Hauser syndrome, aka “congenital absence of uterus and vagina.” Affected individuals much prefer MRKH, because the clinical description is both damaging to self-image and not even accurate. The reproductive tract develops from two parts of the embryo, and only the part that becomes the upper vagina and some of the uterus and cervix are affected.

I’d never heard of MRKH, until a report in the Lancet in April described a tissue-engineered vagina that, being closer to the real thing than anything else so far, can help women with MRKH. And there have been a lot of contenders.

Tissue engineering is a form of regenerative medicine that crafts a replacement part using a patient’s own cells plus synthetic scaffolds and molds. Over time, the cells grow and coax surrounding tissue to partake, as the synthetic materials are resorbed, leaving a functional facsimile of what is missing or had been injured. Of course the patient’s own cells are not rejected.

The very fact that a vagina can be tissue-engineered means that it isn’t just a space, a hole, a nothingness, an absence of something. It is a tubular organ leading from one place to another, like an esophagus or intestine.

Skin, bladders, even nostrils have been fashioned this way. Like the tissue-engineered trachea, a vagina connects the outside to the inside. The laboratory-grown organ hails from the Wake Forest Baptist Medical Center Institute for Regenerative Medicine and the Tissue Engineering Laboratory, Children’s Hospital Mexico, where the pilot clinical trial reported in The Lancet took place.

It is a leap forward from past attempts to fashion a “neovagina” in women with MRKH.

Often a newly-diagnosed teen would be sent home with dilators that resemble test tubes with instructions on how to use them to widen and lengthen the tiny vaginal canal, called a “dimple.” In the best case scenario, a medical team teaches the technique and follows the patient carefully, but sometimes a young woman is left to grapple with the procedure in an embarrassing situation, such as in a college dormitory. Dilation is about 90% successful, and if not, then surgery becomes an option.

An early procedure was the Vecchietti technique, which threads a small plastic orb called an “olive” through the navel, going in and out and down, stretching out a canal to about two finger widths. The McIndoe procedure uses skin from the thigh or buttocks to create a neovagina. Other variations on the theme create a passageway and then line it with all manner of tissue: skin, amniotic membrane, abdominal lining, intestine, inside of the mouth, even cellulose.

None of these substitutes, being monolayers, accurately mimics the lining of a true vagina, which has layers of epithelium, muscles, and connective tissue. That’s where the new neovagina comes in.

Muscle cells are coated onto the substrate (Wake Forest)

Muscle cells are coated onto the substrate (Wake Forest)

It begins with epithelial and muscle cells from the underdeveloped lower vagina. The procedures were done on four young women, from 2005 to 2008, leaving plenty of time for evaluation.

The cells are expanded and seeded onto hand-made biodegradable scaffolds designed to fit each woman’s anatomy. Six weeks after sampling the cells, the tissue-engineered replacements are stitched into a surgically-fashioned canal in the right place. And over time, the body fills in the connective tissue, nerves, and blood vessels, as the synthetic materials melt away.

An MRI shows the tissue-engineered vagina (Wake Forest)

An MRI shows the tissue-engineered vagina (Wake Forest)

The new organs took their places. They had the requisite three tissue layers. They didn’t close up and they work. The patients report normal sexual desire and arousal, lubrication, orgasm, satisfaction, and intercourse without pain.

“This pilot study is the first to demonstrate that vaginal organs can be constructed in the lab and used successfully in humans. This may represent a new option for patients who require vaginal reconstructive surgeries,” said Anthony Atala, MD, director of the program at Wake Forest, in the news release. The researchers didn’t mention sex reassignment surgery, but that’s obviously a possibility.

Although MRKH affects the reproductive tract, women have two X chromosomes and normal external genitalia and secondary sexual characteristics, with no known impact on libido or sexual identity. Fallopian tubes are present. For some women MRKH also brings hearing loss, tinnitus, scoliosis, cardiac problems, fused neck vertebrae, and a kidney that’s abnormal or in the wrong place.

Familial cases of MRKH have been reported as far back as 1888, and a few families with more than one case point to autosomal dominant inheritance. But the highly variable phenotype and inconclusive inheritance patterns suggest a multifactorial etiology.

The Lancet paper on the tissue-engineered vagina led me to a wonderful website, www.beautifulyoumrkh.org, started by PhD geneticist Amy Lossie. One woman writes about doing the exploration that most XXs do when we realize there’s another hole down there, and not finding what she was seeking. She imagines running to her mom yelling, “I can’t find my vagina!” Another recalls a boyfriend who called her a freak.

Language on the website is disturbing: “shame,” “hiding,” “terrible, dark secret.” But the language in the Lancet article makes it understandable: “abnormal,” “defects,” “damage” and “malformations” all appear, some more than once, just in the introduction. It is much worse than being called a default option.

(Wikimedia Commons)

(Wikimedia Commons)


It isn’t easy being different, especially in this most private of parts.
Dr. Lossie told her story in the Huffington Post last year. I recently spoke with her for DNA Science. (My questions in italics.)

Some women on the website report going from doctor to doctor, none recognizing the syndrome — a common experience in the rare disease community. What was yours?

Like most people who have MRKH I just didn’t get my period. I was pretty fortunate because we had a new ob/gyn in my town and he knew what it was right away. I had a diagnosis really early. I didn’t go through a lot of time in doctor’s offices.

How did your feelings change after finding this out about yourself?

In the acute phase of trauma, from 1 to 5 to 10 years, I thought, ‘I’m not who I expected.’ You have to retool your life and look at things from a different perspective. Then, after you do dilation, everyone says you’re ‘fixed.” But I wasn’t fixed.

When I was in my late twenties, everyone started having babies and I couldn’t. You have to figure out how you are going to handle that. For me that was the hardest part.

At the beginning, I had no one to talk to. There were no support groups, there was no Internet yet. You think you are the only person who has this. I spent a lot of time thinking about it by myself. I saw a counselor and went through a grieving process. She gave me permission to grieve for my unborn babies, which helped me accept having MRKH.

Amy C. Lossie, PhD, President and CEO of Beautiful You MRKH Foundation

Amy C. Lossie, PhD, President and CEO of Beautiful You MRKH Foundation

The stories on the website are from all over – India, Australia, Argentina, Norway, Canada. How did you start it?

Meeting other women changed my life. It happened at a retreat in Canada, and the woman who hosted it was becoming a therapist and she used it for her MSW thesis. There were six of us, all about the same age. We shared our stories.

There was a lot of sadness. Hurt, anger. But for 3 days I knew that no one was going to ask me, ‘When are you having a baby?’ No one would ask me about tampons. It was a safe place. For the first time I felt like a normal person. I didn’t have to worry that someone would ask me a question I had to lie about or figure out an answer to. I realized what meeting other women with MRKH meant to start accepting who you are.

We started out creating artwork. The first pieces were how you felt about yourself when you were younger. It was interesting because I’d been journaling for awhile and it hadn’t helped me. But by the end of that weekend people painted beautiful pictures and you could see a transformation. So I started a foundation. I couldn’t not start it.

Around this time, my co-founder, Christina Ruth, had started MRKH Support and Awareness, a Facebook support group for MRKH. I was stunned with the number of women who shared their experiences and sought support on this website. So, I emailed her and asked her to join me. After months of conversations, we realized that we shared common core values for the Beautiful You MRKH Foundation and were very compatible. We had different and complementary strengths that led to a strong partnership.

Our Facebook support group has 800 members, and they know they’re not alone. You can find somebody like you to talk to.

Also see the MRKH Organization Inc. and the Mid-Atlantic MRKH Meet-up Group for info on a meeting June 7 in Philadelphia.

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Great News For The Progeria Community

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(PLoS Biology)

(PLoS Biology)

A repurposed drug that reverses some of the rapid-aging symptoms of Hutchinson-Gilford progeria syndrome also extends life, according to a new report in the journal Circulation. That’s terrific news.

The disease is exceedingly rare, affecting 1 in 4-8 million newborns. Prevalence is 1 in 18 million, reflecting the fact that average lifespan is only 14.6 years.

The effects of the drug on symptoms were announced in fall 2012. I was just starting this blog then, and two of my earliest posts dealt with the disease.
My first post, “Progress for Progeria,” was an interview with Francis Collins, MD, PhD, about how he worked with the founders of the Progeria Research Foundation, Scott Berns, MD and his wife Leslie Gordon, MD, PhD, who were parents to then 22-month-old Sam. He became quite well known in his short time here. He passed away at the age of 17 early this year, and was the subject of the HBO documentary Life According to Sam. Dr. Gordon, from Hasbro Children’s Hospital of Brown University and Boston Children’s Hospital and medical director of the PRF, is first author of the new paper.

The drug lonafarnib alters the morphology (lower right) of the nuclei in cells from  children with progeria. (PLoS Biology)

The drug lonafarnib alters the morphology (lower right) of the nuclear membranes in cells from children with progeria. (PLoS Biology)

The progeria story is a beautiful cascade of discovery.

The work of the PRF led to finding mutations in the lamin A gene that cause progeria, and that revealed the mechanism, which in turn led to realization that a shelved pediatric cancer drug, lonafarnib, targeted the same pathway. Would it work against progeria? My second post, “From Rapid Aging to Common Heart Disease,” chronicled that story.

The short version: A class of drugs called farnesyl transferase inhibitors would remove a small organic molecule, farnesyl, from one end of lamin A protein. The problem behind progeria is that farnesyl groups aren’t removed, as they should be, due to mutation affecting a splice site that would otherwise enable the group to be jettisoned. The result is a version of the protein called progerin.

Normally lamin A forms part of the scaffolding that hugs the inner face of the nuclear membrane, contacting the threads of DNA and their associated proteins (chromatin) in the nucleus. With the farnesyl groups tenaciously hanging on, the altered architecture interferes with the chromatin, a little like poking one’s abdomen and jostling the intestines.

Effects are profound. Progerin impacts DNA replication, RNA transcription, chromatin formation, cell division, apoptosis, and formation of the pores that let molecules in and out of the nucleus. It’s little wonder that a suite of symptoms ensue, the aging connection emerging perhaps as the misshapen nuclear membrane touches the telomeres (chromosome tips), somehow accelerating the shrinkage that marks biological time.

Megan and Devin have progeria. (Progeria Research Foundation)

Megan and Devin have progeria. (Progeria Research Foundation)

The early results reported in 2012 were incremental, yet definitely steps in the right direction. Some children gained weight faster, their arteries grew thinner and more elastic, and their bones strengthened and hearing improved. And now that a few more years have passed, it’s clear that the drug is also extending the short lives of these children and adolescents.

The new study underscores the importance of knowing the natural history of a disease – what happens, when, and for how long. The PRF patient registry identified 204 children, and information on them provided the control information to compare to effects on children given the drug. The researchers also consulted reports in the literature and databases to identify children to match with those being treated.

The trial began in 2007 with 28 children from 13 countries, and at first evaluated only lonafarnib. Two years later the protocol added a statin (pravastatin) and an osteoporosis drug (zoledronate), with funding from PRF and the National Heart, Lung and Blood Institute. These two drugs are also farnesylation inhibitors but complement lonafarnib in action. The investigators hypothesize that the lonafarnib is extending life because of its effects on arteries. The trial grew.



Results reported in the Circulation paper are striking. Among 43 treated children over the 6 years of the study so far, 5 died (11.6%). Among 43 in the “matched comparison group,” 21 died (48.8%).

Mean survival was extended 1.6 years in the treated kids. And that might be an underestimate, because many started the drug when they were far along. Treating earlier might extend survival even further. The researchers estimate that it will take at least 6 more years to confirm the survival benefit.

(Wikimedia Commons)

(Wikimedia Commons)

The numbers are small, the time elapsed short. But an extension of 1.6 years for someone with an average life expectancy of 14 years is impressive.

I very rarely use the word breakthrough. But I’ll make an exception for the continuing success story of slowing down the runaway aging clock that is progeria.

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Mutations in 115-Year-Old Provide Perspective for Personal Genome Sequencing

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A 103-year-old man from Yemen.

A 103-year-old man from Yemen. (wikimedia)

Normally, I wouldn’t post about a report that’s already reverberated through the blogosphere, but the finding of hundreds of mutations in the blood of a 115-year-old woman reminded me of something I’d published a few years ago. In an actual journal. And at least in the accounts I read, no one linked the finding to mutation detection in clinical genome sequencing.

In 2008, Renad I. Zhdanov, a researcher at the Institute of Fundamental Medicine and Biology at the Russian Academy of Sciences, started emailing me, about stem cells. I’d just published a tome for Insight Pharma Reports and a highly forgettable novel on the topic.

397px-Cavities_evolution_1.svgDr. Zhdanov had the idea to use stem cells from the teeth of the oldest old to create spare parts for others. Presumably the cells, having hung around for more than a century, would have exceptional potential. When our discussion veered toward the concept of informed consent — yanking teeth from unsuspecting elders — I realized we could write an editorial for the American Journal of Bioethics, where I had a contact. And so “Centenarians as Stem Cell Donors” appeared in the November 2009 issue. It is, unfortunately, behind a paywall.

Although our article was more whimsical than serious science, we did traipse through the brief history of dental stem cells. NIH researcher Masako Miura discovered them in his 6-year-old’s mouth in 2003, which inspired the spawning of tooth banks, for where there’s a new stem cell, a new company is sure to follow. But saving your kid’s teeth in a jar is just as effective, as my post two weeks ago pointed out for the posthumous diagnosis of Rett syndrome.


A herring a day and OJ were Henny's route to  longevity. (Wikimedia)

A herring a day and OJ were Henny’s route to longevity. (Wikimedia)

The paper in last week’s Genome Research probed a different part of a very old person’s person – the blood cell compartment. It harbors cells that divide frequently and therefore would be most likely to have accumulated lots of mutations, which mostly happen during DNA replication.

Subject “W115,” aka Hendrikje van Andel-Schipper (“Henny”), was born on June 29, 1890 and died on August 30, 2005, living 115 years and 62 days. She was a supercentenarian, older than 110. Currently 72 supercentenarians live in the world, all but 4 of them female. The US has 23. Moses (from the bible, not Gwyneth Paltrow and Chris Martin’s son), who lived to 120, was one.

Despite having had two cancers, Henny never had chemo, and so she was in a sense a mutational virgin. The genetic changes were presumably spontaneous. She spent her final years in a nursing home but was healthy and alert, if frail, to the end. A few days before her death she reportedly told the home’s director, “It’s been nice, but the man upstairs says it’s time to go.

Moses was a supercentenarian. (Wikimedia)

Moses was a supercentenarian. (Wikimedia)

Henny attributed her remarkable longevity to consuming daily herring and orange juice. And the list of conditions she didn’t have was long. Her autopsy revealed no plaques and tangles in her brain, no clogged arteries.

Henne Holstege, PhD, and her colleagues at Vrije University Medical Center in Amsterdam performed whole genome sequencing on white blood cells and brain neurons from Henny’s autopsy. “We compared the genome of peripheral blood cells, derived from hematopoietic stem cells which have experienced many divisions, with the genome of brain cells, which rarely divide after birth. We expected to find mutations in the blood genome but not in the brain genome, and we wanted to assess the type and sites of the mutations,” she wrote in an email. The NIH Director’s Blog explains the connection between Henne and Henny.

(Dept. of Energy)

(Dept. of Energy)

Henny’s white blood cells had some 450 mutations, including 424 single base changes and 22 insertion-deletions (indels), all in nonrepeats. No mutations were found in the brain neurons, nor in the cells of the breast tumor that had been removed when she was 100, nor in the stomach tumor that killed her when it spread.

Even though the mutations weren’t in repeats, they weren’t important enough to have impacted Henny’s survival or health, or so it seems. The mutations were more likely where methyl groups cling to the DNA because it is rich in cytosines and guanines – the “CpG islands” that signify a gene’s beginning and indicate a gene expression pattern more like a stem cell than a specialized cell.

Most of the mutations were not in parts of proteins that algorithms predict would be catastrophic. Nor did they show up in compendiums of cancer mutations (COSMIC) or in the Human Gene Mutation Database. None partook of leukemias, although Henny’s blood cells had variants in some scary cancer genes associated with faulty DNA repair – BRCA1 and 2, RAD50 — but these deviations aren’t associated with disease.

Henny, it appears, was genetically lucky. But the results are also intriguing on the cellular level.


HSCs beget myeloid and lymphoid progenitors, which beget increasingly differentiated blood cells. (Wikimedia)

HSCs beget myeloid and lymphoid progenitors, which beget increasingly differentiated blood cells. (Wikimedia)

White blood cells descend from hematopoietic stem cells (HSCs) from the bone marrow. Mutations happen more in HSCs than in quiescent cells like brain neurons because stem cells divide. They don’t “turn into” anything as the media often oversimplify.

HSCs are the mother cells that top the charts of blood cell lineages that festoon stem cell labs. An adult’s bone marrow has about 11,000 of these plastic cells that can divide to eventually yield almost anything, with about 1,300 HSCs awake enough at any one time to be generating white blood cells, according to a recent study.

Amazingly, most of the mutations in Henny’s blood represent only two active HSCs, one of which was likely the daughter of the other. “The mutations occurred at such frequencies in the peripheral blood that the majority of the blood cells could only be derived from two active hematopoietic stem cells. At first we did not believe that this could be true, but after careful examination there was no other conclusion that we could draw. We speculate that the number of active stem cells may decrease during aging, to the extent that in W115, only two stem cells were active,” Dr. Holstege wrote. Someone did some math and deduced a spontaneous mutation rate of one about every three cell divisions.

Not as surprising, the telomeres (tips) of the chromosomes in the white blood cells were 17 times shorter than the ones from brain cells. Altogether, the scenario suggests what the researchers call “stem cell exhaustion.” Henny had depleted nearly all of her HSCs.



Mutations are generally regarded as bad. I’ve covered several on this blog, from the curious genetics of werewolves to homeotic mutations that turn arms into legs to the sad tales of neurologic disease and hereditary blindness.

But some mutations are good. Perhaps the best is the CCR5 mutation that keeps HIV out of our cells, a genetic glitch that drugs and gene therapy are trying to imitate. Most mutations, it seems, are neither evil nor beneficial, but neutral. After all, Henny lived in good health for 115 years, yet her blood cells still accrued 450 mutations.

DNA Science blog always tries to find a different perspective to genetics news, and for the case of Henny, it is the fact that mutations need not signal doom. Dr. Holstege had the idea to look at Henny’s genome because of the role of somatic mutations in causing cancer. But another view is that many mutations do nothing at all.

Neutral mutations will impact the application of DNA sequencing in health care decision-making. The Nature article featured in last week’s post, “Guidelines for Investigating Causality of Sequence Variants in Human Disease,” for example, outlines how clinical researchers should rank mutations, in terms of the extent of the danger they pose to health or perpetuation of the species. But this week’s paper on mutations in the healthy 115-year-old reiterates that some mutations may have no effect at all. That’s why genome annotation of all possible gene variants and deciphering gene-gene interactions are so important for applying genome information.

As far as mutations go, clearly we’ll have to figure out what’s normal. Results from Henny suggest that to some extent we can embrace our mutations – they are simply a consequence of the changeable nature of DNA. And that is, ultimately, how life began and has evolved.


I’ve just started catching up on Orphan Black, the BBC show about a young woman and her many clones. How were the clones created? Yes, I’m searching for scientific gaffs. Wikipedia says by in vitro fertilization, which makes no sense given meiosis mixing up gene combinations in gametes. I thought of dissolving an 8-celled embryo into 8 individuals, but Sarah already has at least 11 clones. Was it somatic cell nuclear transfer? Then somatic mutations would distinguish the clones. Am I missing something? I’d welcome a guest post on this show!

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Celebrating DNA Day, 2014

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dnaday_Logo2014April 25, DNA Day, commemorates the date in 1953 when James Watson, Francis Crick, Maurice Wilkins, and Rosalind Franklin introduced the structure of DNA in the journal Nature. The anniversary was echoed, if a bit chronologically contrived, half a century later with the publishing of polished human genome sequences.

In celebration of DNA Day 2014, DNA Science blog honors high school students who are thinking and writing about DNA.

I was astonished to discover after my gene therapy book was published two years ago, thanks to a review in School Library Journal, that my target reader was 15 years old. In contrast to the report in this week’s Science that schooling sets back students’ knowledge of evolution, today’s teens know DNA. So here are a trio of looks at DNA-savvy high school students.

Me and March of Dimes essay contest winners: Alexi Ayrey, Molly Ottensoser, Gurjeet Johal, and Jomanda Morales, at NYU 3/25/14

Me and March of Dimes essay contest winners: Alexi Ayrey, Molly Ottensoser, Gurjeet Johal, and Jomanda Morales, at NYU 3/25/14


For the past few weeks I’ve been traveling around New York State for the March of Dimes high school convocation, speaking about children undergoing gene therapy. The program debuted in 1971, with Jonas Salk launching the speaker series. This year I provided the question for an accompanying essay contest:

“It is possible to determine the complete DNA sequence of your genome and identify genes that may cause diseases or indicate your ancestry. Doctors are just now learning how to use the information in a person’s genome. Many genetic diseases do not have treatments. Would you want to know your genome sequence? Which genes would you like to know about, and which not?”

Answers were insightful, optimistic, and proactive, with the students realizing that knowledge of personal genetic information would enable individuals to live in ways that would minimize inherited tendencies towards certain diseases. Yet at the same time, the students recognized limitations of knowing one’s genes.

The winning essay, by Gurjeet Johal, a senior at the High School for Health Professions & Human Services in Manhattan, movingly wrote of her family’s experience with Lewy body dementia. “Having already witnessed the effects of such a disease on my grandfather’s life, I would not want to be informed of my chances of developing it. Even if my gene sequence indicates that I would develop Lewy body dementia, I have nothing within my power to prevent it and awareness would likely cause complications due to the anxiety the discovery would instill in me,” she wrote.


Ms. Johal chose a disease that would fit in perfectly in addressing the question posed in this year’s American Society of Human Genetics DNA Day essay contest:

“Complex traits, such as blood pressure, height, cardiovascular disease, or autism, are the combined result of multiple genes and the environment. For ONE complex human trait of your choosing, identify and explain the contributions of at least one genetic factor AND one environmental factor. How does this interplay lead to a phenotype? Keep in mind that the environment may include nutrition, psychological elements, and other non-genetic factors. If the molecular or biological basis of the interaction between the genetic and environmental factors is known, be sure to discuss it. If not, discuss the gaps in our knowledge of how those factors influence your chosen trait.”

Lewy body dementia is a complex trait – most cases are not inherited, yet mutations in several genes cause familial forms, and variants of other genes contribute to risk. Environmental influences on dementia are not well understood. So it’s possible that Ms. Johal’s risk is not as high as she fears.

The winners of the ASHG contest are Rachel Gleyzer, Adesuwa Ero, and Cameron Springer. Congratulations! “The students submitting the best essays really outdid themselves this year,” said Michael Dougherty, PhD, Director of Education for ASHG. “We continue to be impressed by the quality of their writing and their ability to master some pretty complicated science.”

I read a few of the essays, and they’re quite wonderful. Some “rounded up the usual suspects” among complex traits, such as autism, obesity, and type 2 diabetes, but a few were highly original.

One student chose Huntington disease, which would seem an unlikely candidate for a complex trait because penetrance is close to 100 percent – if you inherit a mutation, you’ll eventually get HD, unless something else gets you first. But due to gene-environment interactions, DNA is never destiny, and this student explored a very subtle manifestation of this interplay — recent findings that diet can influence age of onset of HD. That is empowering information in a traditionally helpless situation.

Another student did the opposite — chose a trait thought of as mostly environmental and discussed the contribution of a single gene: language ability and the FOXP2 gene. “Language owes its potency to its remarkable malleability; it possesses innate grammar encoded in genes and their transcriptional targets, but its phenotypic capacity is still determined by environmental language acquisition,” the student wrote.

A particularly elegant entry parsed possible causes of depression through the lens of being an adolescent. The analysis cited candidate genes, but then discussed effects of sleep deprivation from living linked to our lit devices, coupled with the academic pressures of high school. “Teens predisposed to the disorder because of mutations in genes controlling neurotransmitters may not exhibit any symptoms until confronted with burdening stress.”

The ASHG essays weren’t all gloom and doom. A student wondered why the ability to relate a wavelength of light to perceiving a specific color is taken for granted (color vision), yet the ability to do the same for a sound and a musical note is regarded as a talent (perfect pitch).

Dr. Yuval Itan supervised Benjamin and Mark Mazel last summer as they made the human gene connectome more accessible. (Rockefeller University)

Dr. Yuval Itan supervised Benjamin and Mark Mazel last summer as they made the human gene connectome more accessible. (Rockefeller University)


High school students aren’t only writing about DNA science, they’re doing it.

About a year ago, I posted about the human gene connectome, the physiology-based network that is the brainchild of Rockefeller University postdoctoral researcher Yuval Itan. Two of the co-authors of his new publication at BMC Genomics are in high school, twins Benjamin and Mark Mazel.

“These two very talented students made a web interface for the human gene connectome, which now enables everyone to easily use it. I think that it’s a great example that could give motivation for young students to participate in science and for investigators not to be intimidated by age,” Dr. Itan said.

The connectome uses a “new metric” – a “biological distance” calculated from shared function rather than shared DNA sequence. But originally the database required downloading too much information, inducing what Dr. Itan calls “terminal command line phobia.” So his two young protégées spent last summer applying their computational skills to improving the interface.

Which gene variants are clinically relevant? And for whom? (NHGRI)

Which gene variants are clinically relevant? And for whom? (NHGRI)


Coincidentally, an article published yesterday in Nature ties together the student experiences above: the uncertainty of genetic information that can impact health (March of Dimes), the complexity of many traits (gene:environment interactions; ASHG), and how genes interact (the human gene connectome.)

The Nature paper reports recommendations based on a workshop held at the National Human Genome Research Institute (NHGRI) in September 2012 to discuss ways to assign meaning to gene variants for individuals. It’s all about context.

“Mistakes are happening in the clinic based on questionable evidence of an association. People are jumping to the conclusion that if a patient has the same variant as was previously implicated in a disease, then they must also have the same disease. Medical treatment decisions are then being based on this information, sometimes to the detriment of the patient,” said one of the authors, Teri Manolio, M.D., Ph.D., director of the Division of Genomic Medicine at NHGRI.

I have a broad perspective on the issue from writing and revising my human genetics textbook over the past two decades. And I’m convinced the genetics community has known all along that the human genome sequence itself was only a beginning, despite the hyperbole at the various milestone announcements. Using all of the information in a genome would require understanding not only every gene’s function, but identifying the nuances of every possible variant (base changes and copy numbers), and then the implications of all possible gene-gene and gene-environment interactions. The expectation that knowing the sequence could automatically lead to cures always was a huge oversimplification – a little like reading a novel by speaking each letter aloud, from page 1 until The End, and somehow understanding the story.



Yet it appears that the oversimplification has persisted, judging from a disconnect I sensed in the essays responding to the question I posed. For at the same time that geneticists are rightly warning physicians that incorporating genomics into their practices will not be straightforward, some students think that day is already here. I only read a few of the essays, the top ones, but these ideas emerged:

1. Doctors sequence and interpret genomes. Already. Regularly.
2. There was one human genome project, the government one.
3. All genetic testing stems from the human genome project.
4. Each individual has his or her own genetic code.
5. Gene therapy, including the germline variety, is already being done.

Where are these ideas coming from?

I don’t think it’s from teachers, who only spend a few weeks on genetics and are probably happy just to get through Mendel and DNA structure. A more likely source is the media’s constant barrage of breakthroughs and advances. The uncertainty of using DNA information in diagnosis does not make as compelling a story on the nightly news.

Double Helix with StethoscopeThe difficulty of translating genomics into the clinic IS the story that will ultimately affect most if not all of us. Summed up James Evans, M.D., Ph.D., Bryson Distinguished Professor of Genetics and Medicine at the University of North Carolina at Chapel Hill and co-author of the Nature paper, “Deciding which genomic variants are important players in disease is probably the most difficult challenge that we face in trying to implement genomic data in medicine. It’s difficult to implicate specific variants as having an effect on disease because there are millions of variants in the human genome, and most are rare and do not have a big impact on health. This will likely be a long-term challenge.”

It sure will be, making me wonder what DNA Day will celebrate ten years from now. And twenty years from now.

DNA Day is a terrific tradition. It’s important to acknowledge the past, while realistically projecting where DNA science will take us in the future.

(Many thanks to Mike Dougherty of ASHG for the DNA Day image)

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Chromosomal Clues to Past Pregnancy Loss

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Genetic time stands still within an individual, for the most part.

Genetic time stands still within an individual, for the most part.

Genetics is, in a sense, timeless. DNA sequences stay mostly the same in an individual over a lifetime. Minus the inevitable somatic mutations, the genome of a fertilized ovum is much the same as that of the 80-year-old it might one day become.


My favorite example of genetic time standing still is the three-year-old who died of Rett syndrome in 1991, but wasn’t diagnosed until 2004. A year earlier, her mother had read a magazine article about the syndrome and recognized her daughter’s symptoms: falling, clumsiness, loss of speech, seizures, and the peculiar repetitive hand-wringing characteristic of the disease. Might her daughter be diagnosed posthumously?

The mom had an idea: instead of disinterring the body, could researchers extract DNA from a stored baby tooth? The Australian Rett Syndrome project connected the astute mother with researchers who indeed probed the daughter’s genome in tooth stem cells, finding the telltale mutation in the MECP2 gene.

Diagnosis after death dispelled guilt – the father had blamed himself when his daughter fell down the stairs, and the mother had blamed a vaccination. DNA testing revealed that the mother hadn’t passed on the mutation – it originated in her daughter. And that meant that their other relatives, including the girl’s siblings, weren’t at risk.

The Rett case is a precedent of sorts for “rescue karyotyping” to explain recurrent pregnancy loss, described in a recent paper in Reproductive Biology and Endocrinology, which I summarized for Medscape. In contrast to recent fetal tests, such as non-invasive prenatal testing (NIPT) using cell-free fetal DNA and sequencing fetal genomes, which provide a look forward, rescue karyotyping looks back.

Views_of_a_Foetus_in_the_Womb_detailPREGNANCY LOSS IS COMMON
The birth of a healthy baby is against the biological odds. Of every 100 eggs exposed to sperm, 84 are fertilized, and of these, 69 implant in the uterus. There, 42 survive one week or longer, 37 make it past 6 weeks, and only 31 are born. Of the fertilized ova that cease developing, about half have severe chromosomal abnormalities. The halt comes so early that the event usually goes unnoticed. A late and heavy period.

These odds mean that a miscarriage is a rather common event. Partly for this reason, a couple crushed from their first miscarriage may be comforted, told to try again, and sent home. Devastated.

That may happen with a second miscarriage too. It’s usually at pregnancy loss #3 that a health care provider refers a patient to a genetic counselor, who takes a detailed history and then orders a karyotype – a chromosome check – of the prospective parents. But oftentimes the tests come back with the normal 23 pairs.

The conservative stance in testing after pregnancy loss might be because most chromosomal accidents are just that – an errant chromosome doesn’t part from its homolog and instead follows it along, leading to an egg or sperm with one too many or one too few chromosomes. Because most such “aneuploid” situations are independent events, expensive karyotyping doesn’t make sense, at least not economically. But there are other costs.

“‘We don’t test it. Those are the guidelines.’ That’s what my patients who have had repeat miscarriages tell me. Everyone agrees that you don’t test after the first miscarriage, and most agree not to test even after the second, but to wait for the third,” Zev Williams, M.D., Ph.D., director, Program for Early and Recurrent Pregnancy Loss (PEARL), Montefiore Medical Center/Albert Einstein College of Medicine, recently shared with me. Having had such patients myself as a genetic counselor, seeing them typically after the third loss, I checked. Indeed, the  American College of Obstetrics and Gynecology and the American Society for Reproductive Medicine recommend karyotyping only after the third spontaneous abortion, although a woman’s age or other problems may accelerate that timetable.

Sometimes a more unusual chromosome glitch occurs that can repeat, such as a translocation in which different chromosomes swap parts. The parents would be carriers, but each can make “unbalanced gametes” – eggs or sperm with hunks of genetic material missing or extra. Each conception then faces the not-so-good odds of a chromosomal imbalance that can be incompatible with life or cause birth defects. Knowing about a translocation can be helpful because it recurs with a known frequency, enabling a couple to use technology such as  preimplantation genetic diagnosis to avoid poor outcomes in the future.

Checking chromosomes of the parents is eventually necessary because most women whose pregnancies were once ending did not have the presence of mind to collect and bring a sample of tissue (“products of conception”) to a doctor to send for testing. But if she had a D&C  (dilation-and-curettage) afterwards, a bit of the tragedy may exist on a shelf somewhere, embedded in paraffin. Dr. Williams and his colleagues have gone back to those samples to try to find out why some pregnancies ended.

Hauling out stored samples may seem a low-tech approach in this age of sequencing genomes, but one that can bring peace of mind. For with recurrent pregnancy loss naturally comes guilt.

“Every patient will blame herself. Was it the argument with her husband? Someone smoking nearby? Did she lift something heavy? One woman went on a ski trip and had a miscarriage a few days after and was convinced it was the ski trip. That’s a horrible feeling to have to think that you did something to cause a pregnancy loss,” said Dr. Williams, whose team is questioning 1500 people on the perception and understanding of miscarriage. “It would provide peace of mind to know that it was a trisomy, a triploidy, a tremendous genetic rearrangement and not the stress at work or the fight with the husband. Rescue karyotyping can give a sense of closure to patients who are wracked with guilt,” he explained.



A human karyotype circa mid-1960s would have shown chromosomes of all the same color arranged in groups by size. A child with what was then called mental retardation might have been diagnosed with a “B-group chromosome disorder.”

Karyotyping progressed through ever-more-specific staining, as knowledge of chromosome structure grew, leading to FISH – fluorescence in situ hybridization. FISH uses DNA probes to highlight specific DNA sequences rather than larger-scale structural nuances that affect how dyes bind.

Then came array comparative genomic hybridization (array CGH) and the ability to detect microdeletions and microduplications. This is done during pregnancy and to diagnose children with unexplained developmental delay. But it was the use of array CGH in cancer genetics, on paraffin-embedded tumor samples, that inspired Dr. Williams to retrieve stored tissue from miscarriages past.

“In the cancer field, the push was to do more sensitive testing using higher and higher resolution arrays, to look at small rearrangements. We are looking for higher level anomalies, missing much more, so less stringency is needed. A sample might come back saying ‘insufficient material’ if you want to find a 5 kilobase deletion, but not if it is a question of missing an entire chromosome. That’s easy to answer,” Dr. Williams said. CGH reveals anomalies within that range.


To test the feasibility of rescue karyotyping, the researchers used array CGH on 20 specimens from 17 women who had had recurrent pregnancy loss. Of the four women who’d had fetal chromosomes checked while they were pregnant, three attempts had failed. So rescue karyotyping provided new information on old samples.

Sixteen samples had enough DNA to analyze; the oldest had been stored more than four years. And chromosomal glitches showed up in 8 of the 16: three trisomies (an extra chromosome in all sampled cells), one mosaic trisomy (extra chromosome in some sampled cells), two partial deletions, and two unclassified variants.

As expected, most of the findings indicated a one-time event. But any result is important, Dr. Williams maintains, because of the alleviation of guilt. And the testing seems easy enough to do – once the strategy is validated and standards established, a health care provider would need only find and send tissue blocks to a testing facility.

Karyotyping is a classic technique, perhaps soon to be supplanted by whole genome sequencing, which Dr. Williams and his group and others are already doing. But is that too much information?

“The problem is interpreting the results. All of us have about 2000 mutations. It’s difficult to tell which ones are completely benign. Some might have some advantage, and others might be the cause of a miscarriage. Whole genome sequencing of a fetus will be a difficult route,” he warns.

800px-Sleeping_newborn_infantBut in the meantime, while annotators work furiously to figure out what everything in the genome means, DNA tests on stored products of conception are making past pregnancy losses, for some couples, a little easier to bear.

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Call the Midwife Evokes Cystic Fibrosis in a Simpler Time

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Children with cystic fibrosis too young to pronounce the disease's name call it 65 Roses.

Children with cystic fibrosis too young to pronounce the disease’s name call it 65 Roses.

Previous posts bashing SyFy’s Helix and Dan Brown’s Inferno might suggest that I’m hypercritical of TV and films. Happily, the third season premiere of BBC’s excellent Call the Midwife shows that it is indeed possible to get the science right while telling a compelling story, without special effects and nonsensical intrigue.

On the March 30th episode, a young mother was distraught. Her month-old baby had stopped gaining weight and then started losing it, was fussy and clearly suffering from abdominal pain. He was filling nappies with foul-smelling loose stools so fast that even the father had to help, something unheard of circa 1959 in the poor, working class neighborhood of London’s East End where the series is set.

Nurse/midwife Jenny Lee (Jessica Raine) narrates in flashback the memoirs of the late Jennifer Worth. Not as slick as Downton Abbey, the show presents a glimpse of time that is just as addicting. Nurse Jenny, a 22-year-old of privilege when she arrived at Nonnatus House in 1957, expected to be working at a small private hospital, but instead had to adjust to life in a convent, where the nurse-midwives lived.

The third season opener was a trip back to a time when we didn’t test for dozens of the 1600 mutations known to cause cystic fibrosis (CF), let alone sequence exomes or genomes to get to difficult diagnoses. Instead, observation ruled. And the program did it spectacularly.

Clubbing of fingers reflects poor circulation.

Clubbing of fingers reflects poor circulation.

Many viewers probably recognized the classic signs of CF, but the nurse/midwives, nuns, and lone doctor didn’t. Abdominal pain. Recurrent infections and fevers. Both baby Ian and his toddler brother Martin choked on phlegm. And everyone was deeply puzzled.

Early on, the young father told nurse Jenny that his brother died at age 4, and no one ever knew why. His two young sons were bringing back terrible memories. With the father’s family history quickly uttered, the pieces fell into place, at least in hindsight.

It was astute Sister Monica Joan, whom everyone dismissed as well on the road to dementia, who noted the salty taste to Ian’s brow, ran upstairs to the books that she spent her days meticulously cataloging, and came back down. With a sly smile, she uttered the very quote “from Queen Anne’s time” that is in my and every other genetics textbook:

Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die.

No one paid attention to the silly saying from the 1600s. The young mother blamed herself, as did the male doctor. Perhaps the mother’s depression was making her neglectful, the good doctor, resting and enjoying a smoke, asked as Nurse Jenny exhausted herself thumping on the chest of the baby to free him from the stifling mucus.

Heart-and-lungsA little while later, frustrated at being ignored, the batty old nun braved a rainstorm to thrust the book into the hands of the doubting doc. He finally read it and the light bulb went off. The boys had CF.

Here’s a description of CF from my human genetics textbook (new edition coming in the fall, shameless book plug):

“Physicians first described the condition in medical journals in 1938 as a defect in channels leading from certain glands, causing extremely thick mucus and resulting in infections in the lungs; a clogged pancreas, preventing digestive juices from reaching the intestines; and salty sweat. Children with CF, with their slow growth and frequent infections, are sometimes first diagnosed simply as suffering from ‘failure to thrive.’”

The history of recognizing CF goes back farther. The saying that the nun quoted comes from the German Children’s Songs and Games of Switzerland, and evokes an observation by a Spanish professor of medicine from the early 1600s equating salty skin with being bewitched.

Then in the early 1900s, several physicians noted that oily smelly stools, cough, and death in early childhood often went together. Dr. Dorothy Andersen at Babies’ Hospital of New York published the 1938 paper naming the disease, which describes the problems in the lungs and pancreas.

512px-CFTR_Protein_Panels.svgIn 1953 came the “sweat test,” after a heat wave in New York City filled emergency rooms with kids who had CF. They were the first to dehydrate and suffer from heat exhaustion. In 1989, Drs. Lap-Chee Tsui, Francis Collins, and their colleagues reported discovery of the gene and its encoded protein, the cystic fibrosis transmembrane regulator (CFTR).

The Call the Midwife episode was a true story, that of English actress Jenny Agutter, who plays Sister Julienne. She told writer Heidi Thomas about her own family history.

Agutter lost an older brother Christopher to “unexplained stomach problems,” and remembers, at age 6, looking forward to the homecoming of her new sister Bridget. The baby never left the hospital, and Agutter’s mother never quite recovered from her grief, not understanding that it wasn’t her fault.

In 1980, Agutter’s niece Rachel, daughter of her brother Jonathon, whose childhood was exactly like that of Christopher and the two boys in the episode, was diagnosed. Jenny was tested at age 37 when pregnant and found to be a carrier, but fortunately her husband was not.

The episode ended with the doctor reassuring the parents that things could be done to help their sons. Today’s median life expectancy, the early 40s, is way up from age 5 back in 1938, but not good enough.

Strategies to treat CF have grown more targeted as probing the 1600+ ways that CFTR ion channels misform and misfold has revealed vulnerabilities. Treatments range from classic postural drainage and enzymes sprinkled on food, to antibiotics and anti-inflammatories to prevent and quell infection, to nuclease-based Pulmozyme to break up the sticky mucus. Pulmozyme was the first drug approved solely to treat CF, in 1993.

200px-Fart.svgVibrating vests treat the phenotype; gene therapy has yet to help the genotype, although a drug on the horizon, Ataluren, can shield the nonsense mutations that account for a small percentage of patients, enabling synthesis of functional CFTR protein. One new drug restores the liquid on airway surfaces, and the protein-refolding blockbuster drug Kalydeco refolds the errant chloride channel protein so that it can make its way to the cell membrane, where it establishes the ion flow that keeps secretions moist. Patient chatter on Kalydeco websites reports improvements from amazing strides in lung function to noticeably less stinky farts.

Here’s a pipeline of what’s coming in CF treatment.

But new problems arise. One is multidrug-resistant Mycobacterium abscessus, which is infecting the lungs of up to 10 percent of CF patients in the US and UK. And that may be the tip of an iceberg. Sequencing lung fluid from patients identifies bacterial genomes that we didn’t even know lurk in the lungs ravaged by the disease.

(Dept. of Energy)

(Dept. of Energy)

I loved the episode of Call the Midwife for almost as many reasons as I hated the scientifically illiterate, testosterone-infused Helix and Inferno. The science was accurate, both in historical context and in hindsight. The daily hands-on nurse knew far more than the distant doctor. A woman thought to be demented had a clear memory when it mattered, and noticed a sign of disease that others had missed. And in that little pocket of London poverty decades ago, the women were in charge.

Call the Midwife is a rare gem.

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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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