Exon Skipping: Borrowing from Nature to Treat Rare Genetic Diseases

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

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

Understanding exon skipping requires a short detour into the biology.

(Laurence Hurst et al, PLOS Biology)

(Laurence Hurst et al, PLOS Biology)

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

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

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


February 28

February 28

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

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

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

(Dept. of Energy)

(Dept. of Energy)

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

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

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

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

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

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

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

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

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

Despite these hurdles, the therapy itself is sheer genius.

In DMD, fat gradually replaces muscle.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Insight and application take time.

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Patient-Specific Stem Cells Recapitulate Age-Related Macular Degeneration

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

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

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

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

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

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

iPS cells (Stephen Tsang and colleagues, Columbia University)

iPS cells (Stephen Tsang and colleagues, Columbia University)

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

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

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

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

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

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

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

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

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

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

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

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

Superoxide dismutase (SOD2), our antioxidant enzyme

Superoxide dismutase (SOD2), our antioxidant enzyme

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

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

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

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

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

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

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

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Probing Polar Bodies to Pick Disease-Free Embryos

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

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

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

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

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

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

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

Cannibals, Papua New Guinea

Cannibals, Papua New Guinea

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

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

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

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

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

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

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

February 28

February 28

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Project Prakash: Learning From the Formerly Blind

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Gene Therapy News: Brain, Skin, Eye

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(Jonathan Bailey, NHGRI)

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


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

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

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

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

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

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

A horse virus delivers Parkinson's gene therapy.

A horse virus delivers Parkinson’s gene therapy.

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

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

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

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

Stem cells nestle in the bulge regions of hair follicles.

Stem cells nestle in the bulge regions of hair follicles.

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

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

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

EB blisters the skin.

EB blisters the skin.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Is the mystery virus Ebola?

Is the mystery virus Ebola?

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

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

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

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

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

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

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

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

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

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

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

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

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

Death by Dionne?

Death by Dionne?


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

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

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

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

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

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

(Dept. of Energy)

(Dept. of Energy)


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

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

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

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


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

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

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

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

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

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




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

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

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

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

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

The grossometer ranking for Helix is pretty high.

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

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

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

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



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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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


(Dept. of Energy)

(Dept. of Energy)

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

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

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

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

Yersinia pestis, the cause of the Black Death.

Yersinia pestis, the cause of the Black Death.

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

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

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

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

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


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

But the worst illogic comes towards the end.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Diets That Treat Genetic Disease – Three Classic Cases

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amino acidsThe greatest challenge of majoring in biology in college was mastering the chemical steps that build up and break down the 20 types of amino acids specified by the genetic code. I could memorize the energy pathways with an idiotic mnemonic device – CIA SS F_UCK MY OX – (citrate, isocitrate, α-ketoglutarate, succinyl CoA, succinate, fumarate, malate, oxaloacetate) – and even name the zillions of worms in Zoology 101. But the amino acid pathways really taxed my brain.

Had I known that a glitch in a single such pathway could violently twist a toddler’s body and make her smash her head into a wall, perhaps I would have had the context to appreciate what I was memorizing.

Newborn screening is essential to preventing genetic disease symptoms with dietary intervention. (NHGRI)

Newborn screening is essential to preventing genetic disease symptoms with dietary intervention. (NHGRI)

Breaking down a biological process into chemical steps has made it possible to prevent symptoms of certain inherited “inborn errors of metabolism” with dietary interventions. The key is early detection.

The story of altering “brain nutrition” to treat genetic disease begins with an astute mother who noticed the odd smell of her children’s diapers. The saga continues today at a small clinic in central Pennsylvania where a dedicated pediatrician and his team are applying biochemistry to save children’s lives.

This post continues a series on my two-hour visit to the Clinic for Special Children in Strasburg, Pennsylvania, in early December. (see Part 1: An Advocate For the Amish At A Very Special Clinic and Part 2: Autism, Seizures and the Amish). I detoured on Christmas to make people mad at me with a post on finding a Neanderthal gene variant in modern Latin American populations.

Newborn screening started with stinky diapers.

Newborn screening started with stinky diapers.

In 1931, in Norway, a mother of two young disabled children noted a musty odor to their urine. The father mentioned this to a friend, who told a friend, who was, conveniently, a physician interested in biochemistry. Intrigued, he analyzed the foul urine in a lab at the University of Oslo, with help from the mother, who hauled over bucketfuls of the stuff. The physician, Asbjörn Fölling, identified the problem in the children’s metabolism – later named phenylketonuria (PKU) – and then found it among people languishing in mental institutions. A missing or non-working enzyme blocked their bodies from converting the amino acid phenylalanine into another, tyrosine.

In PKU, excess phenylalanine spills into the urine, the blood, and poisons the brain. In the early 1960s, physician and microbiologist Robert Guthrie, who had PKU in his family, developed what would become known as the “Guthrie test” to detect metabolites in blood (using mass spectrometry) taken from a heelstick shortly after birth.

Because phenylalanine is an amino acid, restricting dietary protein prevents the mental retardation (intellectual disability) of PKU. For a few years, photos of families with PKU showed the eldest children, born pre-diet, sitting in wheelchairs next to their well younger siblings, who’d also inherited PKU but had followed the diet. I had to remove one such photo from my human genetics textbook  when a student recognized her aunt as one of the disabled children. The PKU regimen isn’t something off-the-shelf like gluten-free brownies or low-fat yogurt — it’s a complex “medical food“ that health insurance covers.

A more familiar diet/genetic disease story is that of “Lorenzo’s Oil” to treat adrenoleukodystrophy (ALD), the subject of a 1992 film.

Lorenzo Odone was born in 1978 to Augusto, an economist with the World Bank, and Michaela, a linguist. In summer 1983, Augusto was sent to the Comorros Islands, off the southeastern African coast, and Michaela and Lorenzo came along. It was an idyllic time, Augusto told me in 2010. “Lorenzo learned French and some Comorrian words. He was a very gifted, precocious child.”

When the Odones returned to the United States, Lorenzo started kindergarten, and soon began to have difficulty paying attention. Then he’d throw tantrums and break rules. By the new year he was falling often, and by spring he couldn’t see. Then blackouts and memory loss started. Soon an exhausted Lorenzo could no longer speak, and then seizures began.

After ruling out a brain tumor, epilepsy, Lyme disease, and attention deficit hyperactivity disorder, white spots on a brain MRI indicated ALD. The fatty bubble-wrap-like layers around Lorenzo’s neurons were vanishing, but his parents refused to accept the death-by-age-8 prognosis. Instead, they hit the NIH library in Bethesda, and read about a mother in Norway who’d helped develop the PKU diet.

Michaela and Augusto Odone taught themselves enough biochemistry and nutrition to invent a strategy to attack the defect in fatty acid metabolism that was melting the insulation off their son’s brain cells. They discovered that researchers had already tried to treat ALD by restricting the fatty acids that were building up, but it didn’t work. Then in 1986, researchers found that oleic acid sops up the enzyme needed to make the excess fatty acids. The Odones combined oleic-acid-rich canola, olive, and mustard seed oils to create the eponymous mixture, with the help of a biochemist in England.

Lorenzo’s Oil was no mom-and-pop operation. The Odones worked with prominent researchers and the papers reporting clinical trial results appeared in the Annals of Neurology, The New England Journal of Medicine, and the Journal of the American Medical Association. By the time results were in, Lorenzo’s brain was already too damaged for him to respond to the oil, but he took it anyway. Lorenzo Odone died on May 30, 2008. He choked and then bled to death, possibly because of the blood-thinning effects of the oil. It was the day after his thirtieth birthday.

Did Lorenzo live 23 years longer than expected because of the oil? “It could have been his care and the oil. His mother was very careful, but even so, the oil had something to do with it,” Augosto told me, then paused. “But I’m not sure about that.” He died in October 2013, a few months after his final book, Lorenzo and His Parents, was published. Lorenzo’s disease is now treatable with experimental gene therapy (see chapter 8 in my book on the subject.)

Because of those long-ago college classes, “organic” to me has always meant carbon-containing – not a way to grow vegetables. In the organic acidemias/acidurias, too much of an organic acid is in the blood (“emia”) and/or urine (“uria”). Typically too little of an enzyme required to break down a dietary amino acid leads to build-up of whatever the enzyme normally acts on. These conditions affect metabolism of lysine (the amino acid type that the dinosaurs in Jurassic Park couldn’t make, supposedly ensuring their captivity) and the branched chain amino acids (leucine, isoleucine and valine).

Many of the derangements of organic acids have tongue-twister names, such as 3-hydroxy-3’methylglutaryl-CoA lyase deficiency, but at least one has the graphic moniker “maple syrup urine disease.”

Anyone can get one of these inborn errors – DNA doesn’t know within whom it mutates. But human interactions can concentrate mutations within populations, especially when people carry samples of a larger gene pool to new communities, as happened with the Amish and Mennonites. In this way, mutations rare in the ancestral European gene pool became amplified in North America.

The need to diagnose and treat glutaric aciduria type 1 (GA1) led to the founding of the Clinic For Special Children, as detailed in my first post. It’s the most common single-gene disease in the Amish population that the clinic serves, but until Dr. Holmes Morton began investigating it years ago as a young fellow at Children’s Hospital of Philadelphia, the condition had often been mistaken for cerebral palsy. Children died.

A newborn with GA1 appears well for the first few days, but the urine already has high levels of telltale glutaric acid. Then he or she begins to vomit and refuses to eat. The hallmark of the disease is dystonia, the uncontrollable muscle contractions that cause repetitive twisting and writing. Deterioration of the striatum, in the brain, causes the movement problems. The condition progresses to seizures, which often follow a fever. The throat spasms, scoliosis horrifically bends the back, and the child eventually becomes lethargic and comatose.

Today, the symptoms of GA1 are entirely preventable.



The complexity of the biochemistry behind GA1 makes its dietary treatment even more ingenious than those for PKU and ALD. Because of deficiency of an enzyme (glutaryl-CoA dehydrogenase) lysine isn’t broken down completely, and an intermediate product of the pathway, glutaric acid, accumulates and enters urine and blood.

The first diet to treat GA1, from 1989, restricted protein to lower lysine levels, but it wasn’t very helpful if symptoms were already present. With the advent of newborn screening for GA1 in 1994, affected infants could be identified (often from urine brought in by midwives) before symptoms began.

Several variations on the low-lysine diet theme were tried. Adding an organic acid called L-carnitine sopped up a bit more of the excess glutaric acid, dropping incidence of brain injury from 94 to 36%. But the disease continued on its course.

Clues from fruit bat brains and urine.

A clues from fruit bat brains and urine.

Then Dr. Morton discovered a hint in a 1988 paper about bat urine. Healthy fruit bats pee high levels of glutaric acid, just like kids with GA1, but bats don’t develop neurological symptoms because their brains can process lysine. Could glutaric acid trapped in the brains of children with GA1 be causing their motor symptoms?

In 2005 Dr. Morton deduced, from the bat paper, that any treatment for babies with GA1 had to lower glutaric acid in the brain – not just in the blood or urine. And that led to further study of the breakdown pathway for lysine, but in a very specific circumstance – crossing the blood-brain barrier.

To traverse the tile-like walls of capillaries in the brain, lysine is ferried on a protein called a transporter, but competes with the amino acid arginine to grab a spot. When a baby with GA1 spikes a fever, arginine levels in the blood fall as part of the immune response to infection, and more lysine enters the brain – triggering seizures.

Dr. Morton, Dr. Kevin Strauss, and their colleagues reasoned that a dietary formula that cuts lysine by 50% while doubling arginine might block the transporters from binding too much lysine. The situation is a little like something I discovered at the Atlanta airport a few weeks ago.

My special status with the TSA is like arginine shoving aside lysine on its transporter.

My special status with the TSA is like arginine shoving aside lysine on its transporter.

In 2013, the government investigated a sample of the US population and anointed some of us “low-risk travelers,” harmless enough to breeze through security with our shoes, sweaters, belts and laptops intact and free from interrogation by the Transportation Safety Administration. We special “TSA PreCheck“ folk stream towards the scanners past the regular people, and at the last minute, we’re allowed ahead of them – so I’m like an arginine displacing a lysine as we enter the shared transporter. (Note: my group of TSA-approved travelers were all older white women. Profiling?)

Returning to biochemistry, Drs. Morton and Strauss and their colleagues gave the low lysine/high arginine formula to 6 boys and 6 girls, tracking symptoms and blood glutaric acid levels from 2006 until 2011. Twenty-five children born from 1995 to 2005 who took an earlier recipe served as the control group.



The formula worked, and kids with GA1 who take the medical food grow up healthy. “Over 20 years, GA1 was transformed from an unknown disorder that invariably caused disability and early death to a disorder that is recognized worldwide through newborn screening programs and is routinely treated with good outcomes,” said Dr. Morgan. Newborn screening saves lives.

At the Clinic for Special Children, the squares of a beautiful quilt mounted on a wall represent young patients. One patch, from 1989, has what I thought was a grain silo. But it’s a vial of urine, because “that’s how we did samples for inborn errors back then,” Dr. Morton told me. Today targeted mutation analyses supplement diagnoses based on metabolites, to minimize false positives.

Quilts. Sick children in Amish farmhouses. A mother delivering smelly diapers to a biochemist. Parents teaching themselves fatty acid chemistry to create an oil that would heal their son’s brain.

These low-tech stories of treating genetic disease — from the PKU diet of the 1960s, to Lorenzo’s oil from the 1990s, to the glutaric aciduria story and others that I haven’t yet discussed — illustrate the long evolution of tackling inherited disease, one gene at a time. High-throughput, next-generation, massively-parallel, whatever-the-next-buzzword-is exome and genome sequencing have found their niches in diagnosing novel or “unusual presentations” of inherited disease, but for more than 60 years, new treatments have come largely from an understanding of biochemical pathways and medical sleuthing.

Said Dr. Morton, who has won a Macarthur Award and an Albert Schweitzer Prize for Humanitarianism, “Scientists who work as physicians and care for many patients with the same genetic disorder over long periods of time develop a different understanding of genetic disease than scientists who study disease mechanisms in cell cultures. It is often through the daily work of a physician caring for a patient that new opportunities for treatment are realized.” He calls the daily practice of medicine “the true frontier of translational genetics.”

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Did Mexicans Inherit Diabetes Risk from Neanderthals?

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Did Neanderthals give some of us increased risk for type 2 diabetes? (credit: Knut Finstermeier)

Did Neanderthals give some of us increased risk for type 2 diabetes? (credit: Knut Finstermeier)

These days Neanderthals seem to pop up where you least expect them. When Medscape asked me a few days ago to write up a paper being published in the December 25 online Nature, the title sounded run of the mill: “Sequence variants in SLC16A11 are a common risk factor for type 2 diabetes in Mexico.”

Yawn. Another genome-wide association study (GWAS), showing stretches of single sites in the genome (SNPs) that track with type 2 diabetes in Mexicans and other Latin Americans, who have about twice the prevalence as European whites. I might have been more alert had I read the ending first, like I sometimes do with novels – a “Neanderthal analysis team” that included Svante Paabo. The Neanderthal connection is also not in the headline for the news release accompanying publication of the paper, but is buried a few paragraphs down. So I wondered, on Christmas day, would the media notice the missing link? If they don’t, here’s DNA Science blog’s take.

The study is terrific, with nice numbers — 9.2 million SNPs analyzed for 3,848 Mexicans and other Latin Americans who have type 2 diabetes and 4,366 who don’t. The researchers are a stellar team from Mexico, Boston, LA and others part of the  The Slim Initiative in Genomic Medicine for the Americas (SIGMA) Type 2 Diabetes Consortium.

The findings zeroed in on five linked SNPs — a haplotype — in a gene called SLC16A11 on the short arm of chromosome 17. Four of the five mutations change an amino acid in the encoded protein, and the fifth is silent. The protein normally ferries certain lipids into liver cells, a complex function that makes sense in terms of past studies of insulin resistance. The association between the haplotype and disease risk is strong, and holds up in other populations. Perhaps it could be developed into a tool to predict elevated diabetes risk, or present a new drug target in lipid metabolism. So far, so good.

I'd love to have been a fly on the wall of the Denisovan cave, watching archaic humans interact. (Max Planck Institute for Evolutionary Anthropology)

I’d love to have been a fly on the wall of the Denisovan cave, watching archaic humans swap DNA. (Max Planck Institute for Evolutionary Anthropology)

But it was the DNA sequence that grabbed my full attention, and the clues from geographic prevalence.

The five-site haplotype is in 50% of Native Americans, in about 10% of East Asians, much rarer in Europeans, and absent among Africans. And it’s ancient. Researchers determine the degree to which a mutant gene differs from the most common sequence (wild type), then impose a time scale in the form of  known mutation rates. The SLC16A11 five-site haplotype is so divergent that it goes back to nearly 800,000 years ago — before our ancestors expanded out of Africa.

The most plausible explanation, unexpected I suspect, seemed to be that the haplotype came from an archaic human – a Neanderthal or Denisovan or their as-yet unnamed contemporaries. And the haplotype indeed shows up in the skeleton of a Neanderthal found in the Denisovan cave in Siberia — that’s the now-famous place where the genomes that led to us sorted themselves out. It is a peek into a sometimes promiscuous past.

Having a bit of one’s genome from a Neanderthal that predisposes to diabetes might be unexpected, but the presence of this DNA source shouldn’t be stigmatizing – from 1 to 4 percent of many of our genomes are from Neanderthals (sub-Saharan Africans have none). But for a disease population so scrutinized — note the Pima Indian investigations — it’s surprising to find that a risk gene for diabetes traces back to this side branch from the evolutionary road leading to humanity.

I was going to take a blogging break until this paper appeared — next week I’ll return to the genetic stories of the Amish.

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Autism, Seizures, and the Amish

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(Jonathan Bailey, NHGRI)

(Jonathan Bailey, NHGRI)

On December 4, I visited the Clinic for Special Children in the heart of Pennsylvania Dutch country, where a tiny staff cares for 2000+ patients with a variety of inherited illnesses. Last week’s post described a family in which 5 of 6 children have a seizure disorder that includes autistic features. Investigation of this syndrome over the past 15 years beautifully illustrates the evolution of gene discovery methods before sequencing catapulted us into a new technological age. (Warning: jargon ahead.)

Today, to identify the gene behind an undiagnosed condition in a child, researchers compare the exome (protein-encoding part of a genome) sequences of parents and possibly siblings to identify causative gene variants (alleles). It’s fast.

In the pre-genome era, researchers followed an indirect trajectory to get from phenotype to genotype:

• Noting that symptoms “run in families.”
• Finding the condition to be more common among identical twins than fraternal twins, and among siblings of patients than in the general population.
• Identifying abnormal chromosomes in people with the condition.
• Using genome-wide association studies (GWAS) to identify patterns of genetic variation (at sets of single-base sites called single nucleotide polymorphisms, or SNPs) that might flag a disease-causing variant.
• Identifying the gene and making a mouse model to test treatments.

When the Amish left Switzerland in the early 1700s to escape religious persecution and settled in Pennsylvania, they brought a sampling of the European gene pool. Reproducing among themselves amplified mutations and resulted in “runs of autozygosity” in their genomes – sections of chromosomes that have the same DNA sequences on both copies. Runs of autozygosity indicate that two relatives inherited sets of gene variants from a shared ancestor, such as second cousins from a great-grandparent. These alleles, dubbed “identical by descent” (IDB), make a powerful tool for gene discovery if they show up exclusively in people who have the same disease.

The boy I met at the Clinic and 4 of his 5 siblings have cortical dysplasia-focal epilepsy (CDFE) syndrome. It arises from a single missing DNA base in a gene called CNTNAP2.

Like many tales of gene discovery, the finding that mutations in CNTNAP2 lie behind a variety of brain conditions – autism, seizures, schizophrenia, Tourette syndrome, and language disorders – began with different threads. Let’s focus on the autism connection.

Autism_awareness_ribbon-20051114Even the oldest genetic techniques demonstrate an inherited component to autism. Identical twins are much more likely to both have it than fraternal twins; siblings of a child with autism have a risk 75 times that of the general population.

In 1998, the International Molecular Genetic Study of Autism Consortium used GWAS to identify 6 regions in the genome that tracked with people who have autism – the top contender was on the long arm of the seventh largest chromosome, or “7q.”

Disturbed communication between neurexins and neuroligins may underlie autism. (Rachelbash1 from Wikimedia Commons)

Disturbed communication between neurexins and neuroligins may underlie autism. (Rachelbash1 from Wikimedia Commons)

In 1999, researchers implicated CASPR2, a type of protein called a neurexin that, when abnormal, disrupts sending of a nerve impulse. Neurexins align with other proteins called neuroligins to create the synapses that form as a young child begins to explore the world, consolidating memory into learning.

In 2003 came reports of rearranged chromosomes that disrupt the gene that encodes CASPR2, CNTNAP2, in people with Tourette syndrome and in 2007 with people who have intellectual disability, developmental delay, impaired speech, and hyperactivity, but not Tourette’s. These different conditions aren’t surprising – effects vary depending on where in the brain neurexin levels are imbalanced.

In 2006, researchers at the Clinic For Special Children and the Translational Genomics Research Institute matched a mutation in CNTNAP2 to CDFE syndrome in closely-related Amish kids. Seizures begin at about the age that autistic features tend to emerge — 14 to 20 months.

Before seizures begin, symptoms of CDFE are subtle: minor motor delays, poor deep tendon reflexes, and slightly-large heads. Children have difficulty concentrating, imitating people, and planning movements, such as crawling, cruising, and walking. The seizures are frequent and severe, and their onset heralds the ebbing of skills – language, cognitive, and social. After several years the seizures cease, but intellect remains stalls in childhood and the individual requires lifelong care.

Amish_-_On_the_way_to_school_by_Gadjoboy-cropKevin Strauss, MD, Erik Puffenberger, PhD, and Holmes Morton, MD, from the Clinic and their colleagues used 100,000-SNP microarray devices  to analyze the DNA of four children with CDFE syndrome from three Amish families. They found an autozygous region 7.1 million bases long on the suspected area on 7q. (Today algorithms quickly spot autozygosity in exome sequences.)

The 7.1 million bases include 83 genes, but only a few made sense. The team first sequenced a gene called CENTG3 known to cause other brain disorders. But the sick kids didn’t have mutations in it.

Then Dr. Puffenberger, the geneticist on the team, found a shortcut: he noticed one SNP in the middle of CENTG3 that was heterozygous in two kids (two different variants), rather than homozygous (the same variant in both chromosome copies), marking an end to the identical-by-descent region. “A recombination event in the middle of the gene allowed Eric to get rid of a lot of it to find the mutation. It’s a perfect example of ‘chance favors the prepared mind’,” Dr. Morton told me. That discovery cut the region of interest on 7q to 3.8 million bases.

The second candidate gene, CNTNAP2, straddles the region of interest. Each child was missing a single base from both copies of chromosome 7 there, and each parent had the same mutation, but in only one copy. They’re carriers. It was Mendel’s first law at work.

The team had found their gene. They then looked further in the community, and among 105 healthy Amish people, four were carriers. Nine of 18 patients who had partial seizures but no specific diagnosis, from 7 families, had CDFE syndrome.

The seizures were puzzling. “One mutation can cause different types of seizures. Four kids in one family respond differently. Some are very disabled, some not very affected,” Dr. Morton said. Three kids had surgery to relieve the seizures, but relief didn’t last. However, the surgeries supplied samples of brain tissue that enabled the researchers to better describe what was going wrong.

Connectivity in the seizure-prone brains is a mess. Gray matter and white matter boundaries blur, and some parts of the cerebral cortex are thickened. The neurons themselves aren’t quite right. They’re too round, too tightly packed, with deranged dendritic trees. Dots on the neurons hint at too many nuclei from glia, the supportive cells that comprise most of the nervous system. The 2006 research report waxed poetic, describing the amygdala, the seat of emotions, in the epileptic brains as “eccentric microscopic islands of partially matured neuronal precursors in tight clusters” cloaked in glia run amok.

The portrait of the Amish epileptic brain made sense in light of the work on the neurexin protein CASPR2 (which stands for contactin-associated protein-like 2). The neurexin forms a scaffolding at the nodes of Ranvier. The nodes are the exposed sites on an axon between pillows of  myelin, the insulating material that is actually the cell membranes of glia wrapped around the neuron like a bandaid around a finger. Nerve impulses hurdle the nodes, sending messages fast enough to sustain life.

The CASPR2 proteins in the Amish kids are stunted. They do not traverse the neuron cell membranes and dip into the cytoplasm as they should, and as a result nearby potassium channels collapse. These channels normally allow potassium ions to rush out of the nerve cells as an impulse passes, resetting it. So without the neurexin scaffolding, the neuron can’t reset itself. Transmission halts. And, somehow, seizures begin. I don’t think it’s known whether the seizures induce the autistic features or they arise directly – further genetic studies should indicate that.

Daniel Geschwind, MD, PhD and professor of neurology at the David Geffen School of Medicine at UCLA, was working on autism genes, and read the 2006 paper in the New England Journal of Medicine. “He called and said, ‘you found my gene!’ A nice collaboration began, and he made a mouse with the Amish mutation,” said Dr. Morton. The mice have the CNTNAP2 gene knocked out, and like people, they have seizures and autistic features.

“A mouse usually runs the around cage, normally social and chattering. These mice were neither,” Dr. Morton explained. The mice also displayed repetitive behaviors and had seizures.

The brains of the mutant mice showed an abnormal connectivity pattern reminiscent of the earlier histology work. “The front of the brain talks mostly with itself. It doesn’t communicate as much with other parts of the brain and lacks long-range connections to the back of the brain,” Dr. Geschwind said. The group had shown similar abnormalities in the brains of children with autism.

Functional MRI shows distinct and consistent connectivity patterns in the brains of children with autism and the CNTNAP2 risk variant. (Geschwind lab)

Functional MRI shows distinct and consistent connectivity patterns in the brains of children with autism and the CNTNAP2 risk variant. (Geschwind lab)

The striking similarity between the Amish children and the mice provides a testing ground for drugs. Risperidone, prescribed to treat repetitive behaviors in children, had the same effect on the mice, while also improving their nest-building ability. But the drug doesn’t help children socialize.

An obvious candidate drug to improve social skills is the “love hormone” oxytocin. It’s abundant in the same brain neurons that are rich in CASPR2 protein. Could too little oxytocin cause autistic features? Results of supplementing oxytocin are promising, both in mice and children.

Dr. Geschwind and co-workers found that a nasal spray of oxytocin “dramatically improves social deficits” in the mice. Because non-autistic mice didn’t respond, the hormone indeed seems to compensate for a deficit.

Amish farmers already give oxytocin to cows to contract their uterine muscles, and I recall getting it to rev up a stalled labor. But don’t try this at home. Several clinical trials are underway for oxytocin or drugs that boost its activity in the brain to improve socialization in children with autism.

Autism Speaks funded the first clinical trial of oxytocin in 2010, and NIH is sponsoring a larger ongoing trial of oxytocin nasal spray. But as far as I can tell, patients were enrolled based on clinical diagnoses according to the DSM-IV – not the more specific criterion of genotype.

Although the idea to try oxytocin to improve social symptoms in autism didn’t require knowing the underlying mutation, such information can add precision to any conclusions by considering mechanism – which can lead to developing other treatments. In another post I’ll look at how genetic precision enabled Dr. Morton to develop cures for certain inborn errors of metabolism that are much more common among the Amish, but still show up on newborn screens of everyone.

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