Getting to the Bottom of Fecal Transplants

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Fecal bacteria sampleImagine that you are a bacterium, comfortably living inside a human bowel movement.

Suddenly, a chemical attack kills most of your neighbors. As other types of microorganisms arrive and begin to take over the vacated niches, they alter the milieu so that you’re washed out in a sudden stream propelled by a blast of gas. How can your few surviving colleagues back in the colon re-establish the peaceful old community?

An infusion of feces from another body can reboot a healthy microbiome in the large intestine (colon), in a biological gentrification of sorts that’s been well studied and much discussed. Now, Vincent B. Young and his team from the University of Michigan and the Essentia Institute of Rural Health in Duluth report in the May/June issue of mBio the biological functions that “fecal microbiota transplantation” (FMT) alters to restore the neighborhood of the colon.


Clostridium difficile (USDA)

Clostridium difficile can take over when antibiotics cleanse the intestines (NHGRI)

FMT delivers other peoples’ excrement to treat recalcitrant infections of Clostridium difficile, a painful and sometimes lethal condition that sweeps in after antibiotics have altered the gut microbiome. In recent years ”C. diff” infection incidence and severity have been on the rise.

Fecal transplants have been done in cattle (via enema) for a century, and on people, in various settings, since the late 1950s. Marie Myung-Ok Lee’s “Why I Donated My Stool,” in the The New York Times a year ago, traces the approach even farther back. She recounts a DIY experience, doctor-guided, that indeed helped her friend with ulcerative colitis. And the New England Journal of Medicine published the straight poop last year demonstrating efficacy.

Feces are a very accessible research material chock full of bacteria. Along the 5 feet of loops of the colon live some 6,800 bacterial species. In one of the first microbiome studies (the subject of one of my very first blog posts and the classic example I use in my textbook), researchers chronicled the establishment of the gut bacterial community by tracking the contents of soiled diapers from 14 healthy babies for the first year, one the child of the chief investigator.

Studies aren't necessary to distinguish between the BMs of breast vs bottle fed infants -- it's obvious.

Studies aren’t necessary to demonstrate microbiome differences between breast vs bottle fed infants — it’s obvious.

David Relman, Patrick Brown and their colleagues at Stanford University, today a powerhouse of microbiome research, found that the babies’ bacteria were quite different at the outset, but by the end of the year, their communities resembled those in the adult digestive tract. And it was published right here at PLOS.

(I ventured briefly into the realm of the microbiome for Medscape, reporting on distinctions between the circumcised and uncircumcised penile ecosystems.)

In the new study, 14 people who’d suffered at least two C. difficile infections received FMT. And it was, as metagenomic studies tend to be, a tremendously data-rich endeavour.

But before I get to the results, let’s address the product and its delivery system. I usually skim, skip, or read last the Methods section of a paper, but in this case I read it first. Just out of curiosity. And it instantly convinced me that my recent decision to switch from the drip coffee method to a French press was wise.

“Donor stool … was collected 6 hours prior to the procedure and then brought to the clinic for preparation of the stool suspension by laboratory staff. The stool was then combined with 90 ml sterile saline and processed in a blender until a smooth consistency is reached. The suspension was then filtered using a coffee filter twice, yielding 40 to 60 ml of stool suspension to be used for transplantation.”

The product, delivered through a nasogastric tube, looks like a melted frozen coffee drink.



The processing destroys the distinctive morphology of feces as depicted so colorfully in the Bristol Stool Chart, a medical tool that I will readily admit I had not heard of. (You can order a coffee mug festooned with the chart.) Ken Heaton, from the University of Bristol, invented it in 1997. Apparently the presentations of human turds hold clues to digestive health.

The researchers identified the bacterial residents in feces from the 14 participants, before and after treatment, from ribosomal RNA sequences, a tried-and-true way to tell eukaryotes (us) from prokaryotes (them). (No fancy genome sequencing required.) Overall, Bacteroidetes become more abundant while Proteobacteria become less so as new feces take up residence.

But the new investigation also imputed what was going on metabolically – presumably so that one day these exact effects can be mimicked by some more palatable approach. “If we can understand the functions that are missing, we can identify supplemental bacteria or chemicals that could be given therapeutically to help restore proper gut function,” Dr. Young said. It reminds me a little of developing infant formula by trying to recreate human milk.

Enterococcus fecalis, a normal resident of the human colon. (NHGRI)

Enterococcus faecalis, a normal resident of the human colon. (USDA)

The analytical tools used offer quite a data dump. Software called “mothur” identifies “operational taxonomic units” (OTUs), which I assume are something akin to species. Then to get at what these microbes are doing rather than simply what they are, the researchers used HUMAnN (HMP Unified Metabolic Analysis Network), which taps into such resources as the KEGG (Kyoto Encyclopedia of Genes and Genomes). Then something called PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) provides the “metagenomics contribution,” the acronym evoking the image of a “meadow muffin,” one of my favorite scatological synonyms.

Put another way, what, exactly, does the new crap do?

The analysis found 75 “gene modules” of 5 to 20 genes each. And their functions at first conjured up bad memories of graduate school courses in biochemistry. Things that change as new bacteria move in include:

amino acid synthesis and degradation
pace of the citric acid cycle
function of amino acyl tRNA synthetases (key enzymes in protein synthesis)
vitamin and nucleic acid metabolism

Many altered activities were classed as “environmental information processing,” which I deduced from the details referred to a lot of schlepping of amino acids and sugars.

Spermidine contributes some odor to stools. (Wikimedia)

Spermidine contributes some odor to stools. (Wikimedia)

Also altered pre- and post-transplant were levels of spermidine and putrescine,” “foul-smelling organic compounds” initially isolated from rotting meat and semen, respectively. They produce odors reminiscent of rotting flesh, halitosis, and, despite the name, the piscine-like scent of a vaginal bacterial infection.

Some biochemical pathways that didn’t work well in the throes of a bout with C. diff recovered after the treatment. Other pathways revved up after treatment, such as changes in glutamate and gamma amino butyric acid (GABA) metabolism that indicate stressed bacteria.

But remembering biochem isn’t necessary to follow the terrific mBio paper, because a beautifully clear figure lists the pathways on the left, and color-coded sets of three horizontal bars on the right: red for “pre-FMT,” green for “post-FMT,” and blue for the donor material. The green bars inch along from red to blue as the microbial community recovers.

The study confirmed efficacy. Five of the 14 participants still tested positive for C. diff after treatment, but 3 of them were clinically okay, the fourth improved on vancomycin, and the fifth was lost to follow up when the study ended at 6 months. That’s a 12/14 or 86% success rate.

Perhaps one shouldn't mess with a functioning microbiome. (NHGRI)

Perhaps one shouldn’t mess with a functioning microbiome. (NHGRI)

“The bottom line is fecal transplants work, and not by just supplying a missing bug but a missing function being carried out by multiple organisms in the transplanted feces,” Young said. “By restoring this function, C. difficile isn’t allowed to grow unchecked, and the whole ecosystem is able to recover.”

The treatment brings back “colonization resistance,” which is the ability to fend off pathogens that comes with the natural gut microbiome. All of this confirms my long-held hypothesis that bowel-cleansing regimens make little biological sense. Leave nature be.

In May 2013 the Food and Drug Administration announced that it would regulate FMT as an  investigational new drug, but a public hearing led to loosening of that requirement.

Discussion continues about whether human feces for transplant should be regulated as a drug or as a tissue. Meanwhile, stool banks have been established, procedures are being performed in hospitals to treat C. difficile infections, and I’m sure companies are exploring the potential new market. I ventured into a health food supermarket today just to be sure they aren’t jumping the gun, and to my relief, among the gas suppressors and bowel cleansers, I didn’t find anything resembling stool replacement. I suspect the approach may have a bit of a PR problem, a little like comandeering HIV to deliver gene therapy.

Dr. Young and colleagues call for further research to better define the risks of fecal transplants: viral or bacterial infection or inflammatory bowel disease exacerbation in the short-term, and the effects of replacing the gut microbiome with a “non-self” set of microbes in the long term.

I hope we won’t be seeing excrement elixirs as dinnertime infomercials just yet.

(opening photo courtesy of University of Minnesota, via Wikimedia)

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Catching Up With 3 Rare Disease Families

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"If you hear hoofbeats, think horses, not zebras," goes the medical mantra. Rare diseases are unicorns.

If you hear hoofbeats, think horses, not zebras,” goes the medical mantra. The 7,000 rare diseases are unicorns.

Four-year-old Eliza O’Neill’s viral videos, the subject of my last two blog posts, continue to dominate the news media with another appearance on The Today Show June 17. Hopefully, her family’s fight to fund gene therapy for her rare disease, Sanfilippo syndrome type A, will focus more attention on the entire rare disease community – 30 million people in the U.S. alone.

That’s a lot of families.

Four years ago, I spent the summer getting to know the families whose stories became my gene therapy book. Thanks to social media we’ve stayed in touch, and I’ve met many others. All continue to astonish me. Here’s a catch-up with three families featured in past posts.

Laura King Edwards ran the Thunder Road half marathon blindfolded, in honor of her sister Taylor. Beside her is Dr. Steve Gray, PI of gene therapy trials for two brain diseases.

Laura King Edwards ran the Thunder Road half marathon blindfolded, in honor of her sister Taylor. Beside her is Dr. Steve Gray, PI of gene therapy trials for two brain diseases.

Laura King Edwards posted at DNA Science a year ago about her younger sister Taylor, now 15, who was diagnosed with ceroid lipofuscinosis, neuronal type 1 – aka Batten disease – when she was 7. Recalls Laura:

“In the worst hour of our lives, we learned that my bright-eyed, golden-haired, intelligent sister – a second grader who loved to sing and dance and run and play – would go blind, have seizures, and lose the ability to walk, talk, and swallow food. She would deteriorate … confined to a wheelchair. She would have to have a feeding tube. Eventually, she would die – blind, bedridden, and unable to communicate.”

Laura eloquently captures her sister’s life and her family’s efforts to help fund a gene therapy clinical trial at her blog, Write the Happy Ending. A post from last week is particularly heartbreaking. Rather than charting her sister’s decline with brain scans or mobility tests, Laura notes that in the 6 weeks between haircuts, Taylor lost the ability to walk. Last week, she had to be carried up the stairs to the hairdresser. This week, she’s in the hospital.

To better get into her sister’s head, Laura runs races blindfolded.

“I do the runs for a variety of reasons. I’ve always been a runner, and running helped me face Taylor’s illness when she was first diagnosed. After watching her run the first of two 5Ks with her Girls on the Run team despite battling Batten disease (and she was already blind at that point), I started running in her honor. I mainly run for Taylor to raise awareness, but my runs have also raised money for Taylor’s Tale. The Thunder Road half marathon I ran with Dr. Steve Gray in November raised money for the (gene therapy) project at the University of North Carolina.

I’ve run 18 races for Taylor. Thunder Road was the only race I ran blind, but I went on 18 blind training runs to get ready for it.

635205790291677074During my months of training to become a blind runner and far more so in the months following the race, my sister slipped farther down the chasm of Batten disease. It is a deep, dark chasm. There are no footholds for climbing out, and some days, no light reaches her ledge. And yet, each day she teaches me something new about courage; each day, she imparts some great piece of wisdom without having to say anything at all.

My next challenge is to run a race in all 50 states for Taylor to continue spreading awareness of Batten disease and build support for the rare disease community. I’m kicking it off this summer!”


Ten-year-old Hannah Sames also has a very rare inherited disease of the nervous system, giant axonal neuropathy (GAN). DNA Science told her story about a year ago too.

In GAN, intermediate filaments composed of a protein called gigaxonin overgrow and run askew, hampering nerve function. Hannah is very slowly losing mobility, and suffers from kidney stones and visual loss, as the lack of gigaxonin in various body parts makes its presence known in ebbing motor and sensory functions.

Dr. Gray (behind Laura in the photo above) began working on gene therapy for GAN before he took on the Batten disease project, and the GAN trial is set to begin within the next few months at the NIH Clinical Center. The trial is largely possible due to the constant networking, meeting-holding, and fundraising efforts of Hannah’s family – parents Lori and Matt, and sisters Reagan and Madison. Their Hannah’s Hope Fund (HHF) was born in the days following the diagnosis in 2008. The highlight is the annual ball, held in February in snowy Albany, NY, near the Sames (and my) home. From Lori:

Doris Buffett's Sunshine Lady Foundation donated $500,000 in matching funds to Hannah's Hope Fund for GAN.

Doris Buffett’s Sunshine Lady Foundation donated $500,000 in matching funds to Hannah’s Hope Fund for GAN.

“The Hope and Love Ball began 5 years ago when friends, Todd and Beth Silaika and Tim and Lee Wilson, approached us with the idea. The first formal gala in 2010 netted $90,000 and was a Valentine theme, fitting for February. Other themes followed: Monte Carlo, Mardi Gras, Midnight in Paris, and Candyland this year, which netted more than $165,000.

In 2010, HHF was awarded a $500,000 all-or-nothing matching challenge grant from Doris Buffett’s Sunshine Lady Foundation. The deadline to raise the funds was the night of the Ball. Snow kept Ms. Buffett (Warren’s sister) away the evening when more than 450 HHF supporters celebrated the success of the $1.2 million, 6-month “Hope for a Million” fundraising campaign. Ms. Buffett was the highlight of the event the following year.

To date, HHF has raised $6 million in 6 years, grassroots, with the vast majority of funds spent on the GAN gene delivery Investigational New Drug (IND) work. The FDA placed the protocol on “Active” status at the end of May, awaiting IRB approval of the GAN gene delivery system. Then trial recruitment can begin.

Unfortunately, Hannah, the inspiration of HHF, has a homozygous deletion mutation. She isn’t a candidate for the phase 1 trial because only missense mutation patients will initially be included. Hannah is awaiting the results of a non-human primate study aimed at inducing tolerance to an intracellular transgene in the CNS. If tolerance is achieved, it will likely be 10 months to a year before Hannah can receive gene delivery.”

(Hannah doesn’t make gigaxonin at all, and so introducing it into her spinal cord, via healthy genes in viral vectors, could trigger an explosive immune response. The other kids who will be in the trial make abnormal forms of the protein, and so their immune systems are already alerted that gigaxonin is a “self” protein.)


Michael and Mitchell Smedley and their friends brainstormed the Bike the Basin event.

Michael and Mitchell Smedley and their friends brainstormed the Bike the Basin event.

A few months ago at DNA Science, Kristen Smedley told how she and her husband Mike assembled a research team to pursue gene therapy for the CRB1 form of Leber congenital amaurosis, which has robbed their sons Michael and Mitchell of sight.

But the boys are more interested in having fun than recruiting researchers, so they dreamed up the hugely successful Bike the Basin event, a half-mile race at the Northampton Civic Center Basin in Bucks County, PA. Kristen continues.

“Back in summer 2011 when the Curing Retinal Blindness Foundation launched, I asked my kids to come up with a fundraiser that could get their friends involved and start getting the word out about our big mission. I wanted my boys to take the lead because while it’s nice that so many people want to help them due to their blindness, my guys need to be able to show the world that they can help themselves.

We gathered about 15 of their closest friends at my kitchen table and the boys pitched their idea of a bike event fundraiser. The kids brainstormed ideas of how to make it work (with parents taking notes and serving lots of ice cream) and Bike the Basin was born!

Just under three months later, the first event raised $20,000. The first three BTB events raised just over $200,000 combined, and the goal for 2014 (Oct 5th) is $250,000. We’ve raised about $80K so far!”


Hannah and her sisters and parents.

Hannah and her sisters and parents.

The families who raise funds for gene therapy clinical trials begin with their own relatives in mind and perhaps as a way to channel their anxiety and fear into something productive. But their generosity extends much farther.

As rare disease-based communities form and strengthen, certain individuals emerge as catalysts. Laura King Edwards, Lori Sames, and Kristen Smedley are three.

Gene therapy will almost certainly be too late for Taylor, and possibly for Hannah. But the Smedley boys may one day be able to see. And Eliza O’Neill may find her way into a clinical trial before Sanfilippo syndrome darkens her sunny childhood, thanks to the efforts of the media to share her story, and the kindness of so many strangers. But Eliza is one child, representing one unicorn. There are so many more.

Whatever the future holds, the efforts of these brave families will reverberate for years to come, measured in the numbers of lives improved or saved.

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Eliza’s Journey: Part 2

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Gene therapy is, finally, about to take off! The feeling was palpable at the American Society of Gene and Cell Therapy annual meeting a few weeks ago. It’s been nearly a quarter century since the first experiment in humans.

The drug development pipeline is beginning to swell with early-stage trials, while a few candidates are closing in on FDA approval for marketing. And just as the annual meeting got underway, Francis Collins, director of the NIH, announced his approval of the Institute of Medicine’s late-2013 recommendation to eliminate certain redundant regulatory hurdles in the launch of a clinical trial of gene transfer. That’s what the technology is technically termed before showing efficacy. It’s a way to deliver healthy genes to compensate for those affected by a particular disease.

There’s more encouraging news.

Spark Therapeutics is preparing to commerical gene therapies for Leber congenital amaurosis type 2 and hemophilia B. (courtesy Spark Therapeutics).

Spark Therapeutics is preparing to commercialize gene therapies for Leber congenital amaurosis type 2 and hemophilia B. (courtesy Spark Therapeutics).

In May, Spark Therapeutics, affiliated with the Children’s Hospital of Philadelphia Center for Cellular and Molecular Therapeutics, announced $72.8 million in new funding, with efforts initially in RPE65-related blindness and factor IX deficiency (hemophilia B). Both indications are far along the development trajectory.

Searching for “gene therapy” at brings up 3,547 hits, but that includes terminated and completed projects. Gene Therapy Clinical Trials Worldwide, in the
Journal of Gene Medicine, lists the number of ongoing trials as 1,991. And gene therapy came up several times two days ago during testimony to the House Energy and Commerce Committees about promoting “21st century cures.”

And still more …

The problem of gene therapy strategies that vanquished inherited disease but caused leukemia is disappearing, thanks to retooled or replaced viral vectors (a future post) and tremendous cooperation among research groups in several nations. And my latest jobs list from Linked/In included several biotech/pharma companies seeking experts in regulatory affairs for gene therapies.

Perhaps most important in the evolution of gene therapy are the contributions from families of kids with rare genetic diseases, many of whom have shared their stories on this blog.

Last week’s post introduced the O’Neills, whose viral video has raised more than $1 million towards launching a phase 1/2 clinical trial (safety and possibly efficacy) for gene transfer to treat Sanfilippo syndrome type A. Without intervention, in the next year or two, 4-year-old Eliza will begin to descend into the nightmare of irreversible brain damage as an enzyme deficiency upsets the biochemical balance in her cells’ lysosomes, the debris sacs.

The media, in droves, conveyed Eliza’s need in early April. Most reported that research so far is only in mice, perhaps because results from a clinical trial in France weren’t officially published until May, in Human Gene Therapy, a source that might be off the radar of many media folk.

The French company,, founded by Sanfilippo parents in 2009, delivered genes by catheter directly into the brains of four children. Results were in by June 2013, a month before Eliza was diagnosed. The trial that she may participate in at Nationwide Children’s Hospital in Columbus, Ohio, will deliver the needed genes much less invasively, by intravenous infusion. The two trials use different viral vectors.

Michaël Hocquemiller, PhD, who handles scientific and clinical affairs at Lysogene, shared good news and links: to last month’s publication, FDA’s granting of orphan drug designation to SAF-301 a year ago, and recent financing. Their procedure appears to be safe, but the trial couldn’t show much benefit in so few children over so short a time. Still, “neuropsychological evaluation revealed an improvement of behavior: a strong decrease in hyperactivity, strongly improved sleeping patterns and improved focus and socialization skills in several patients,” he said via email.

Safety in a few kids is a giant first step, and it’s why additional trials are imperative to confirm the findings and demonstrate efficacy.

I haven’t yet spoken to the researchers at Nationwide, but a news release tells how families helped to fund and participated in the natural history studies that are critical to setting parameters to assess whether a gene transfer protocol is working. The project may begin a clinical trial by the end of this year, with a boost in funding vector production from the efforts of the O’Neill family and the generosity of so many caring strangers.

The story of how the family’s video came to be is in itself fascinating. So here is Part 2 of Eliza’s Journey, from Glenn O’Neill.

SavingEliza - instagramWe knew we had to jump right in. We started our own non-profit 501c3 Cure Sanfilippo Foundation (Tax ID: 46-4322131) with no paid employees and all net funding to the cause currently going toward supporting this clinical trial.

Through March we had raised $250,000 from traditional fundraising we continue today. During this time, we kept thinking we would catch a break and meet an “angel” donor, or celebrity, or corporation who would bail us out. It hadn’t happened and we’d only received a long list of declines.

Early in February 2014, I realized our fundraising at this pace wouldn’t get us anywhere close to the goal. How could we get more people to hear Eliza’s story? We had exhausted our Facebook, email, and social media friends. We were out of options.

One very late night in February, out of desperation, I did a simple Google search for “how to make a viral video.” I sent a “shot in the dark” email to the writer of the first article that came up: Karen Cheng – Give it 100.

She responded!

dnaKaren reached out to a few of her friends on our behalf, and long story short, in the last week of March 2014, freelance videographer Benjamin Von Wong and 2 other artists stayed at our house for 8 days, all sleeping on couches. They filmed 40 hours of footage, editing along the way, and charged us nothing! They bonded with our kids and my wife and I joked that it reminded us a bit of our college dorm life. We had the first real laughter since the diagnosis.

When they left, they gave us the 3-minute video of our very personal story, which does a better job explaining the time-critical situation Eliza is facing far better than any words written here could.

This video was released on April 2nd and went viral the following week. Since then, the video has raised over $820,000 with over 16,500 donors. We are closing in on the Most Ever Raised on, which is just over $800,000.

The SavingEliza video has had more than 270,000 views and the video featuring Eliza’s brother Beckham is approaching 100,000 views. Fox News, ABC News, NBC News, the Today Show, MSNBC Live, Al Jazeera America, Huffington Post, BBC, and many others have covered Eliza’s story. (These figures have now been dwarfed. A third video documents the making of the one that went viral.)

Our fundraising had just been catapulted to a completely new level, and honestly, we weren’t ready for it. There had been no real business plan in place for this wild idea and it all happened so quickly. We scrambled for help from friends and family with the increased administration that comes with this. But what we did have now was renewed energy, renewed hope! While there is still a long way to go on funding, we truly felt for the first time that perhaps money would not be a limiting factor. Perhaps we could get there.

In the next weeks, our foundation will be funding the vector production for Sanfilippo Type A, to be used in the clinical trial at Nationwide Children’s Hospital. This was made possible by the kindness of complete strangers around the world, $10, $20, $50 at a time. This is a crucial and time-sensitive step as it takes 6 months to produce these gene therapy viral vector doses.

The O'Neills

The O’Neills

We move now to The Final Step, which is funding the actual clinical trial. We look to raise over $1M more by October 2014.

The amazing researchers at Nationwide Children’s Hospital have been working more than 15 years to get to a point where this disease can be treated. So many incredible families have funded much of this research throughout the years, and we owe them our deepest gratitude. We need this final push now so never again does a parent have to hear “Sanfilippo syndrome” followed by the words “no cure and no treatment.” And never again does a child have to suffer the devastating effects of this disease. Just this week, another beautiful little girl, age 10, is in hospice and her family is making arrangements. It has to stop.

We’ve received so much positive feedback, many with inspirational phrases like “keep going,” “never stop,” and “we are all with you.” We’re in the midst of a historic new model where social media is funding research. Where everyday people, like you and me, from around the world are helping with any amount they are able, to stop this deadly childhood disease this year….and to save Eliza!

Looking ahead, thanks to the help of so many caring strangers.

Looking ahead, thanks to the help of so many caring strangers.

I go back to my journal entry from July 17 of last year, and those words still apply, but in a much different way than they did then. My goal is still to keep Eliza “happy and smiling,” but now for her, it can last a full lifetime.

(Thanks to Glenn O’Neill for family photos)

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Eliza’s Journey: Part 1

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Eliza O'Neill has San Filippo syndrome.

Eliza O’Neill has Sanfilippo syndrome.

If ten science writers were asked to write a book about gene therapy, a biotechnology with roots going back to the 1950s, they could tell ten different stories.

Any account of gene therapy would include the first experiment in humans, on a 4-year-old in 1990. The narrative  would tell of the dual tragedies that until recently defined the field: 18-year-old Jesse Gelsinger, whose immune system lethally rejected treatment for a urea cycle disorder in 1999, and then the young boys who, shortly after, developed leukemia following successful treatment of their inherited immune deficiency. The cancer hadn’t happened in the mice used to clear the gene transfer protocol for clinical trials.

After revisiting the tragedies, my book The Forever Fix: Gene Therapy and the Boy Who Saved It introduces adrenoleukodystrophy (ALD) to showcase family activism, giant axonal neuropathy (GAN) to tell what it takes to get a gene therapy clinical trial up and running, and then Canavan disease to see what happens years after gene therapy. Sandwiching those cases is the experience of Corey Haas, who would surely be blind if not for gene therapy in 2008, when he was 8.

But I could have told, instead, other ongoing stories of the evolving gene therapy — hemophilia Bbattling Batten disease, Wiskott-Aldrich syndrome, metachromatic leukodystrophy, cancers or HIV … I cover some of them here at DNA Science, and at Medscape Medical News, as the successes accrue. It’s finally happening.

Corey Haas, who had gene therapy to treat Leber congenital amaurosis type 2, would never be able to fish in the Hudson near his home had he not had gene therapy. (Nancy Haas)

Corey Haas, who had gene therapy to treat Leber congenital amaurosis type 2, would never be able to fish in the Hudson near his home had he not had gene therapy. By now he would have been blind. (Nancy Haas)

I recently realized, though, after learning of 4-year-old Eliza O’Neill’s future with Sanfilippo syndrome, that The Forever Fix only touched on two types of experiences.

The lucky, like Corey, found gene therapy because a physician knew about a clinical trial, and they fit the criteria. The many more unlucky are the parents who receive a deadly diagnosis and then discover that no one is pursuing treatment. It’s up to them.

The families are a key part of many gene therapy advances. For ALD it’s a pioneering trio of sisters (Amber Salzman, Rachel Salzman, and Eve Lapin). And the soon-to-start gene therapy clinical trial for GAN is largely possible thanks to the Herculean efforts of Lori and Matt Sames. (Their beautiful daughter Hannah, now 10, graces the cover of the new edition of my human genetics textbook, not for the neural destruction of GAN, but for her unusually  kinky curls, also part of the phenotype.)

Exquisite little Eliza represents a third group: a gene therapy clinical trial nearly ready to go that runs out of money. Creating, selecting, and scaling up the viral vector that delivers the healing genes is expensive. Still, Eliza’s parents, Cara and Glenn, didn’t have to start from scratch like the Sames’ and the Salzman sisters. They and other Sanfilippo families are desperately trying to fund the making of the medicine that is gene therapy.

Racing against a rapid killer must go beyond golf tournaments and bake sales, road races and dances, and other classic fundraising routes that work when one has more time. The families need to raise about $2.5 million, and soon. Thanks to a video (Saving Eliza) that went viral in April and the media appearances that followed, donations are coming in. But social media are ephemeral, and the amount raised isn’t enough, yet.


(Dept. of Energy)

(Dept. of Energy)

Gene therapy introduces working copies of a gene that is absent or malfunctioning in a disease, usually aboard a virus or encased in fatty bubbles. Technically it’s “gene transfer” until evidence shows that it works.

The gene therapy for Sanfilippo syndrome uses a viral vector that enters the brain, meaning that the treatment is a simple infusion – not the skull-piercing catheters once used to deliver genes to children who have Canavan disease.

Sanfilippo syndrome is a lysosomal storage disease (LSD). The lysosomes are the “suicide sacs” of the cell, each housing 43 types of enzymes that dismantle specific molecules. A mutation that robs a cell of just one of these enzymes sets into motion an LSD.

Some LSDs, like Tay-Sachs disease, have telltale signs even before birth, if one could look, in addition to the detectable enzyme deficiency. The molecule that the enzyme should destroy builds up, as the molecule that would result from its action ebbs away, like a knot in a filled garden hose causing a backup at one end and a dribble at the other. In Sanfilippo syndrome, aka mucopolysaccharidosis (MPS) type IIIA, the missing enzyme is heparin sulfate sulfatase.

Eliza and Beckham (Glenn O'Neill)

Eliza and Beckham (Glenn O’Neill)

Eliza’s 7-year-old brother Beckham describes his sister’s “very bad disease” more clearly than I just did, in this second video: “It clogs up her brain and that makes her not learn very well. She’s hyper.” The condition is autosomal recessive, inherited from two carrier parents. Therefore each child has a one in four chance of sharing Eliza’s fate.

So here, from Glenn O’Neill, is the first part of Eliza’s story.

We found out the terrible news today. For now, I want to focus on her wonderful personality and life every day. One of my goals is to keep her happy and smiling for as long as possible. I love her so much.

This was my journal entry on the evening of July 17th, 2013. I never kept a journal before this. Earlier that day, our 4-year-old daughter Eliza was diagnosed with a rare terminal genetic disease called Sanfilippo syndrome type A. In one terrifying instant, we were told that we would have to watch Eliza fade away before our eyes. My journal entry words reflected the lack of hope a parent first feels when told their child has a disease that has no cure and no treatment.

Eliza and other children with this disease are missing an essential enzyme for normal cellular function. Over time, a toxic material called heparin sulfate builds up in their brain and body, leading to severe disability and death before they even reach their teens. This disease affects both genders, all races, all countries and continents. It is rare, but it is everywhere and the world needs to know.

Right now Eliza is a fun-loving 4-year-old who loves to talk, sing, run and MOST of all, cuddle. She loves to play dress-up and horse around with her rowdy big brother Beckham, who fortunately does not have Sanfilippo. She is, however, beginning to show signs of the disease in her learning and attention. And if nothing changes, it will only get worse. And quickly.

By age 6, most children with her disease have irreversible brain damage and lose the ability to speak. As the disease continues to tear through her brain and body, she will lose the ability to walk and eventually she won’t even be able to feed herself. Seizures and painful movement disorders will take over. Life expectancy is usually early teens but preceded by this severe disability.

These devastating changes are a 100% certainty if she doesn’t get treated, and soon. It is a parent’s worst nightmare, and an unfair sentence for any innocent child.

After diagnosis, we quickly began looking for researchers working on Sanfilippo, here in the US and around the world. There weren’t many. My wife Cara, who is a pediatrician for special needs children (ironically), made some key contacts who pointed us in the right directions.

What we found was amazing.

The O'Neills

The O’Neills

What we found was HOPE, and near term! There is a gene therapy trial being scheduled for late 2014 that is specifically for children with Sanfilippo syndrome types A and B, the two most common forms of the disease.

Researchers at Nationwide Children’s Hospital in Columbus, Ohio, have shown that the gene therapy stopped the disease in animal models. It is a one-time injection, delivering the gene that encodes the enzyme missing in the disease using the vector AAV9, which has the ability to cross the blood-brain barrier and clear the storage.

This treatment could save Eliza. In addition, this delivery method, if successful, has great possibility to be used in other MPS disorders as well as more common neurological diseases.

One of the main things standing between Eliza and her miracle is money. The trial is lacking funding to make the medicine, administer the treatment, and remain on schedule. Every moment counts as Eliza approaches the tipping point when her disease will take an irreversible turn for the worst.

The total amount needed to fund both production of the medicine (gene therapy viral vector) and conducting the clinical trial for Sanfilippo type A is $2.5 million. What would you do if you knew that money was the only thing standing between your child and her chance at a full and happy life? What would any parent do?

We could not just stand by and watch our little girl lose everything she is, suffer unimaginable pain and frustration and ultimately die. What would we tell her big brother in a few years, when the disease has taken over completely? What would we tell ourselves?

Glenn O’Neill

Eliza’s Journey will continue next week with Glenn’s description of their campaign, which brought Eliza’s plight before the world. I’ll follow up with more on the science. In the meantime, please help. I learned from chatter last week at the American Society of Gene and Cell Therapy annual meeting that one reason why Europe leads the way in gene therapy is that funding comes largely from charity. That means US.
Twitter: @SavingEliza #SavingEliza
Checks can be sent to: Cure Sanfilippo Foundation, PO Box 6901, Columbia, SC 29260

Thanks to Glenn O’Neill for family photos.

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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,, 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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