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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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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