Phoenix in Mythology and Sequencing

  • Like a Phoenix, Helicos Sequencing is Being Reborn
  • Direct Genomics in China to Launch the Genocare Clinical Sequencer
  • SeqLL in the USA to Launch Benchtop tSMS Sequencer

A phoenix as depicted by F.J. Bertuch (1747–1822). Taken from Wikipedia.org

In ancient Greek mythology, a phoenix is a bird that is cyclically regenerated or reborn by arising from the ashes of its predecessor, which dies in a show of flames and combustion. In contrast to a phoenix, modern biotech methods generally “die” in utility by being displaced with faster, better, and/or cheaper methods rather than undergoing “rebirth” in the context of a new application. However, a method developed by a company named Helicos (scarily close to Helios associate with a phoenix) may prove to be a rare exception. Perhaps this is destiny, but I digress…

Helicos Sequencing

Successful Sanger sequencing of a human genome in the early 2000s spawned numerous efforts to develop faster, better, and/or cheaper methodology to enable genomic analysis on a routine basis. Among the early contenders there was Helicos BioSciences, which was founded in 2003 by several principals including then—and still—uber-famous Stephen Quake.

Helicos sequencing technology, which is depicted below and outlined elsewhere, was especially attractive because it was “true” single-molecule sequencing (i.e. sample prep did not require prior PCR or other amplification, thus greatly simplifying the workflow). Moreover, the technology uniquely allowed direct RNA sequencing, thus obviating the need to first convert RNA into cDNA.

Main steps for primer(P)-based, single-color (Cy3 dye) Helicos sequencing, in this example using two passes. Taken from Harris et al. Science (2008)

3’-Unblocked reversible terminator. Taken from Chen et al. (2013)

Details for how this sequencing-by-synthesis occurs can read in various proof-of-concept publications. However, it’s worth noting here that the 3’-unblocked reversible terminator nucleotide triphosphate monomers have a cleavable linker attached to a detectable dye. Helicos referred to these as “Virtual Terminator” nucleotides since they are efficiently incorporated by a polymerase yet block incorporation of a second nucleotide on a homopolymer template.

So, with these methodological advantages going for it, why did Helicos file for bankruptcy in 2012? Press coverage at that time stated ‘rough financial sledding and tough competition from rival next-generation sequencing companies.’ In my humble opinion, this lack of commercial success was primarily due to the HeliScope Genetic Analysis System (pictured below) being way too big (think upright freezer-refrigerator), far too expensive ($1,350,000), and its ~35-base reads too short on performance—pun intended.

Two Phoenix-Like Versions of Helicos Sequencing

Fast forwarding about five years from the 2012 bankruptcy filing by Helicos brings us to recent reports of two independent efforts to bring back Helicos sequencing in commercially viable formats and contexts, think Phoenix rising from the ashes.

Jiankui He and the GenoCare sequencer (credit Xinjie Tian). Taken from Cyranoski Nature Biotechnology (2016)

The first of these is led by Jiankui He, Founder/CEO of Direct Genomics in Shenzhen, Guangdong, China, as well as Associate Professor at South University of Science and Technology of China in Shenzhen. He, coincidentally, was a postdoc with Helicos cofounder Stephen Quake, who is reported to lead the scientific advisory board for this new company.

The company’s website homepage states the following:

“Direct Genomics is providing physicians with the first single molecule sequencer built exclusively for the clinic. The technology simplifies genome sequencing by reading individual DNA and RNA molecules directly from patient’s blood or tissue samples, which delivers significant improvement in cost and speed. Together with clinicians, Direct Genomics is making genetics an affordable part of everyday patient care.”

Perusal of scant technical information on the company’s website suggests to me that a smaller sized, TIRF-optics-enabled instrument running Helicos-type sequencing has been developed. A story about Direct Genomics by David Cyranoski in Nature Biotechnology states that $100 “clinical sequencing” is being targeted, with a blood-draw to report turnaround time of 20 hours. A very recent publication I found provides details for resequencing the Escherichia coli genome by the Direct genomics platform named GenoCare.

The company’s website lists the following clinical applications:

  • Non-invasive prenatal testing (NIPT)
  • Tumor diagnosis
  • Early-stage cancer prediction
  • Pre-implantation genetic diagnosis (PGD)

The second Phoenix-like rebirth of Helicos sequencing has been developed by SeqLL, which was co-founded in 2013 by William St. Laurent and Daniel Jones, who previously held various technical positions at Helicos. Statements and a video on SeqLL’s website indicate to me that the sequencing technology is essentially that originally developed and patented by Helicos, which is still trademarked as True Single Molecule Sequencing (tSMS™).

William St. Laurent. /   Daniel Jones. Taken seqll.com

SeqLL has been operating as a tSMS™ service provider, but in October 2016 announced the launch of the tSMS™ System Early Access Program giving researchers access to its new benchtop system “designed to deliver unparalleled quantitative RNA and specialty DNA sequencing results to both academic and industry research partners.” I should add that a big, strong bench is needed given that the physical specs are 30 x 30 x 60 inches and 1,000 pounds! Nevertheless, SeqLL recently announced an SBIG grant for improving its direct RNA sequencing technology, which I think could prove to be a driver for adoption.

In conclusion, I think it’s very interesting to see Helicos sequencing coming back to life, if you will, in not one but two different commercial contexts, both of which will hopefully be successful. This despite current ‘tough competition from rival next-generation sequencing companies,’ as observed in the 2012 bankruptcy story about Helicos mentioned above. First and foremost, among that competition is Oxford Nanopore, which I’ve blogged about previously, and whom offers single-molecule sequencing that seems to me to be faster, better, and cheaper for both DNA and RNA, directly.

As usual, your comments are welcomed.

Postscript

After this blog was written, it was reported in GenomeWeb that Direct Genomics plans to deliver 50 instruments this year to SinoTech Genomics, a startup based in Shanghai that offers both clinical and research sequencing services. Direct Genomics CEO Jiankui He is quoted as saying that ‘SinoTech Genomics [is] committed to ultimately purchasing 700 GenoCare platforms,’ and that Direct Genomics ‘has the capacity of producing around 1,000 GenoCare instruments per year,’ which would be very impressive based on past operational experience with manufacturing Sanger sequencers at ABI.

The piece went on to report that Direct Genomics also ‘aims to launch GenoCare in the US in September.’ Regarding what’s inside the box, so to speak, ABI veteran Bill Efcavitch, who previously served as chief technology officer of Helicos, is quoted as saying that ‘the main difference between the former Helicos technology and the GenoCare platform is in the hardware. It’s completely different engineering.’ He added, however, that it still uses Helicos’ virtual terminator chemistry.

Ocean ‘Dandruff’ DNA to Better Study Marine Biology

  • DNA Barcoding for all Organisms has Numerous Applications
  • DNA Barcodes from Water Samples Greatly Aide Marine Biologists
  • Aquatic Environmental DNA (eDNA) Proves to be Informative ‘Dandruff’

Human DNA identity analysis is now commonplace methodology that’s frequently featured in newspaper stories, TV crime series, or “who dun it” movies. The same principle (i.e. using a characteristic DNA pattern or signature) applies to identification of all animals, birds, insects, and microbes. Actually, DNA barcoding extends to any organism, whether it is alive or has been dead for hundreds of thousands of years (so long as it’s preserved by fossilization).

Taken from gajitz.com

Marine biologists face a serious challenge with accounting for very diverse forms of marine life that exists in a mindboggling huge volume of water. Consequently, it’s not surprising that analysis of water-borne, marine DNA barcodes—as proxies for going to and counting fish—is rapidly trending in utility and importance. Known formally as environmental DNA (eDNA), the aquatic version has been humorously referred to as ocean ‘dandruff’ by Christopher Jerde of the University of Nevada in Reno (which, ironically, is landlocked and distant from any ocean.) But I digress. Before diving further (pun intended) into ocean dandruff, let’s briefly review the background of DNA barcoding.

DNA Barcodes 101

Prof. Paul Herbert. Taken from uoguelph.ca

In 2003, Prof. Paul Herbert and coworkers in the Department of Zoology at the University of Guelph in Canada published a seminal study titled Barcoding animal life: cytochrome c oxidase subunit 1 (CO1) divergences among closely related species that fundamentally changed the field of taxonomy. In a nutshell, Herbert’s team showed it was feasible to classify millions of species based only on DNA sequence of the mitochondrial gene CO1. In the intervening, relatively short amount of time, there have been thousands of publications dealing with applications and extensions of this concept, which is now recognized to be very powerful and promising albeit with some limitations.

Typically, DNA barcodes are identified by sequencing after PCR amplification of one or more specific genetic loci such as CO1. Following proof that a DNA barcode can differentiate the species of interest, single- or multiplex quantitative PCR (qPCR) can be used to enumerate relative amounts of sample from the field.

The advent of high-throughput sequencing technologies applicable to complex mixtures of individually tagged samples then gave rise to “metabarcoding,” about which interested readers can consult many publications for specific topics.

Craig Venter steers his research yacht, Sorcerer II, under the Sydney Harbour Bridge in his quest to collect microbes from the world’s waters. Photo: Dallas Kilponen. Taken from smh.com.au

BTW, among the many pioneering scientific ventures by uber-famous Craig Venter, is his Global Ocean Sampling Expedition aboard his research yacht, Sorcerer II. The expedition is a quest to unlock the secrets of the oceans by sampling, sequencing and metabarcoding DNA of all (or most) microorganisms living in these waters.

Lest you think this was a well-intended but unproductive journey—some say junket—by Venter and coworkers, here’s a link to peruse 16 resultant publications that I found by searching PubMed. To watch and listen to Venter talk about this work, you can click here for an educational and entertaining—as usual with Venter—TED Talk on Sampling the Ocean’s DNA that’s had over 550,000 views!

Ocean ‘Dandruff’

Now that we’ve covered the basics of DNA barcoding and metabarcoding, let’s turn back to ocean dandruff. Dandruff, simply put, is dead skin cells. Using dandruff as an intended witty metaphor for ocean eDNA is a bit misleading as marine eDNA is comprised of a complex mixture of cellular matter from scales, feces, decomposing tissue, etc. of fish and all other present or past sea creatures. Consequently, the design and specificity of primers for PCR is of paramount importance for obtaining—let alone interpreting—DNA barcodes based on fragment size or sequence.

As reported by Miya et al., monitoring the occurrence of fish species-specific eDNA PCR fragments (~70–300 bp) has traditionally used conventional electrophoretic gel separation and detection. More recently, qPCR using fluorogenic probes has been employed owing to the method’s sensitivity, specificity and potential to quantify the target DNA. For example, it has been possible to accurately estimate the biomass of common carp in a natural freshwater lagoon using qPCR of eDNA concentrations and biomass in aquaria and experimental ponds.

Miya et al. also describe the development of a set of PCR primers for metabarcoding mitochondrial DNA of 880 species of fish. They sampled eDNA from four tanks with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing. Out of the 180 marine fish species contained in the four tanks, they detected 168 species (93.3%) distributed across 59 families and 123 genera. That’s quite an impressive accomplishment.

Ocean Dandruff Case Studies

Since there are so many fish-related applications of DNA barcodes, I’ve selected several recent examples that are indicative of the utility of ocean ‘dandruff’—and are quite interesting, in my opinion. The first case in point exemplifies how eDNA can be used to deal with rare and endangered species, which are either very hard to find or can be dangerously distressed by catching to obtain samples.

Green SturgeonBergman et al. report that a decline in abundance of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning by these Green Sturgeon are effective at monitoring fish densities in concentrated pool habitats, results do not scale well—pun intended. By contrast, eDNA provides a relatively quick, inexpensive tool to efficiently identify and monitor Green Sturgeon DNA.

Taken from mthsecology.wikispaces.com

These investigators concluded that follow-on work based on this first-ever eDNA study of Green Sturgeon has the potential to provide better knowledge of the spatial extent of Green Sturgeon spawning that could help identify previously unknown spawning habitats and discover factors influencing habitat usage, guiding future conservation efforts.

Monterey Bay—The second case study, by Port et al., involves taking stock of the marine mammals and fish in Monterey Bay using eDNA and, importantly, comparing the results obtained to those from traditional dive surveys.

In brief, this team of researchers from several universities and the Monterey Bay Aquarium Research Institute found that eDNA assessments picked up almost all the organisms scuba divers spied underwater—plus many more that human eyes missed. Here’s some detail on how they did this.

At each scuba survey location as well as at sites offshore, ~1 gallon of water was sampled several feet above the bottom. Four types of habitats were sampled: sea grass beds, Monterey Bay’s unique “Kelp Forest,” sandy areas and rocky reefs. Onshore, in a “clean” (DNA-free) lab, these water samples were filtered to collect cells containing eDNA for storage at −80 °C until eDNA extraction at a university clean lab. A vertebrate‐specific primer set targeting a small region of the mitochondrial DNA 12S rRNA gene was used for PCR followed by gel purification.

Researchers collecting water in Monterey Bay for eDNA analysis. Courtesy Jesse Port. Taken from mercurynews.com

After quantification, pooled amplicons (each having a sample index sequence) were paired-end sequenced on the Illumina MiSeq platform using a 20% PhiX spike‐in control to improve the quality of low‐diversity samples. The conclusions are worth quoting because—in my opinion—the findings represent a new era in marine biology based on nucleic acid analysis:

“We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 meters. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.”

Restated more simply, eDNA analysis of the water picked up 11 of the 12 fish and marine mammals that the divers observed, and—importantly—identified 18 additional animals the divers missed! The efficiency and improvement offered by eDNA analysis compared to traditional seek-and-count methods has been echoed in an editorial I found by Hoffmann et al. titled, tongue-in-cheek, Aquatic biodiversity assessment for the lazy.

Invasive Gobies—The third and final case study deals with detection of invasive, non-native fish to assess whether eDNA can provide a better advanced warning system for detecting these unwanted creatures and implementing eradication steps.

Gobies are an invasive fish species that has colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby (Neogobius melanostomus), pictured below, is among the worst invaders in Europe. Current methods to detect the presence of these gobies are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile.

Taken from animal.memozee.com

To improve monitoring, Swiss and Canadian collaborators developed an assay based on the detection of eDNA in river water, without detecting any native fish species, which is obviously an important assay criterion. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by novices and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s reports and fish community monitoring.

I could go on and on with examples of utility and the many advantages provided by eDNA for marine biology, but I’m sure you get the picture. I hope that you agree with me that eDNA analysis is a very valuable type of trending nucleic acid-based methodology.

As usual, your thoughts or comments are welcomed.

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You and Your Microbiome – Part 3

  • Top 10 Cited Microbiome Publications are Summarized
  • Welcome to the New World-View of “Holobionts”
  • TriLink Products Cited in Numerous Microbiome Publications

It’s been almost two-and-a-half years since posting Part 2 in this series on microbiomes, which I first began in 2013, and the publication rate keeps accelerating, with about 7,000 articles indexed in PubMed in 2016—way more than the mere 35 in 1996. This vast amount of new microbiome information being published annually led me to use the following search strategy to guide my selection of what’s trending in importance for microbiomes.

Basically, I used Google Scholar to search for publications since 2015 that had the term “microbiome” in the title and, among those items found, used the number of citations as a quantitative indicator of interest, importance, and/or impact. But before summarizing my findings for these Top 10 Most Cited Microbiome articles, here’s what you can read in my previous two postings on microbiomes in case you missed them or want to refresh your memory:

Proportion of cells in the human body. You are comprised of much more than what you think you are! Taken from amnh.org

Meet Your Microbiome: The Other Part of You

  • What’s in your microbiome? Why does it matter?
  • Next-generation sequencing is revealing that you and your bacterial microbiome have a biological relationship.

You and Your Microbiome – Part 2

  • Global obesity epidemic is linked to gut microbiome.
  • Investments in microbiome-based therapies are increasing.

Top 10 Cited Microbiome Publications 

The following articles, which were all published in 2015, are listed in decreasing order of the number of citations in Google Scholar. Titles are linked to original documents for interested readers to consult, and synopses represent my attempt to capture essential findings.

1. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile (369 citations)

C. difficile (From lactobacto.com)

Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens such as Clostridium difficile, which is a major cause of antibiotic-induced diarrhea in hospitalized patients. It was found that Clostridium scindens, a bile acid 7-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration as a probiotic, enhances resistance to C. difficile infection.

2. Dynamics and stabilization of the human gut microbiome during the first year of life (298 citations)

Applying metagenomic sequencing analysis on fecal samples from a large cohort of Swedish infants and their mothers, the gut microbiome during the first year of life was characterized to assess the impact of mode of delivery and feeding. In contrast to vaginally delivered infants, the gut microbiota of infants delivered by C-section showed significantly less resemblance to their mothers. Nutrition had a major impact on early microbiota composition and function, with cessation of breast-feeding, rather than introduction of solid food, being required for maturation into an adult-like microbiota.

Graphical abstract by Bäckhed et al. Cell Host & Microbe (2015)

3. Structure and function of the global ocean microbiome (238 citations)

Taken from Sunagawa et al. Science (2015)

Metagenomic sequencing data from 243 ocean samples from 68 locations across the globe was used to generate an ocean microbial reference gene catalog with >40 million novel sequences from viruses, prokaryotes, and picoeukaryotes. This ocean microbial core community has 73% of its abundance shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

4. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis (200 citations)

Taken from factvsfitness.com

The brain-gut axis is a bidirectional communication system between the central nervous system and the gastrointestinal tract. Serotonin functions as a key neurotransmitter at both terminals of this network. Accumulating evidence points to a critical role for the gut microbiome in regulating normal functioning of this axis. The developing serotonergic system may be vulnerable to differential microbial colonization patterns prior to the emergence of a stable adult-like gut microbiota. At the other extreme of life, the decreased diversity and stability of the gut microbiota may dictate serotonin-related health problems in the elderly. Therapeutic targeting of the gut microbiota might be a viable treatment strategy for serotonin-related brain-gut axis disorders.

5. The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes (184 citations)

Taken from dtc.ucsf.edu

Colonization of the fetal and infant gut microbiome results in dynamic changes in diversity, which can impact disease susceptibility. To examine the relationship between human gut microbiome dynamics throughout infancy and type 1 diabetes (T1D), a cohort of 33 infants genetically predisposed to type 1 diabetes (T1D) was examined to model trajectories of microbial abundances through infancy. A marked drop in diversity was observed in T1D progressors in the time window between seroconversion and T1D diagnosis, accompanied by spikes in inflammation-favoring organisms, gene functions, and serum and stool metabolites. These trends in the human infant gut microbiome thus distinguish T1D progressors from nonprogressors.

6. The microbiome of uncontacted Amerindians (150 citations)

Taken from robertharding.com

Sequencing of fecal, oral, and skin bacterial samples was used to characterize microbiomes and antibiotic resistance genes (resistome) of members of an isolated Yanomami Amerindian village in the Amazon with no documented previous contact with Western people. These Yanomami harbor a microbiome with the highest diversity of bacteria and genetic functions ever reported in a human group. Despite their isolation, presumably for >11,000 years since their ancestors arrived in South America, and no known exposure to antibiotics, they harbor bacteria that carry functional antibiotic resistance (AR) genes, including those that confer resistance to synthetic antibiotics. These results suggest that westernization significantly affects human microbiome diversity and that functional AR genes appear to be a feature of the human microbiome even in the absence of exposure to commercial antibiotics.

7. Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology (136 citations)

Taken from dtc.ucsf.edu

Individuals with obesity and type 2 diabetes differ from lean and healthy individuals in their abundance of certain gut microbial species and microbial gene richness. This study in humans found that, at baseline, A. muciniphila was inversely related to fasting glucose, waist-to-hip ratio and subcutaneous adipocyte diameter. Subjects with higher gene richness and A. muciniphila abundance exhibited the healthiest metabolic status. Individuals with higher baseline A. muciniphila displayed greater improvement in insulin sensitivity markers and other clinical parameters. A. muciniphila is therefore associated with a healthier metabolic status and better clinical outcomes for overweight/obese adults.

8. Host biology in light of the microbiome: ten principles of holobionts and hologenomes (132 citations)

Today, animals and plants are no longer viewed as autonomous entities, but rather as “holobionts“, composed of the host plus all of its symbiotic microbes. The term “holobiont” refers to symbiotic associations throughout a significant portion of an organism’s lifetime, with the prefix holo- derived from the Greek word holos, meaning whole or entire. Holobiont is now generally used to mean every macrobe and its numerous microbial associates, and the term importantly fills the gap in what to call such assemblages. Symbiotic microbes are fundamental to nearly every aspect of host form, function, and fitness, including traits that once seemed intangible to microbiology: behavior, sociality, and the origin of species. Microbiology thus has a central role of in the life sciences, as opposed to a “bit part.”

Taken from researchgate.net

9. The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development (131 citations)

The nasopharynx (NP) is a reservoir for microbes associated with acute
respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked
to asthma development. The NP microbiome examination during the first year of life in a cohort of 234 children led to characterization of viral and bacterial communities, and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns.

10. Insights into the role of the microbiome in obesity and type 2 diabetes (128 citations)

Obesity and type 2 diabetes (T2D) are associated with changes in the composition of the intestinal microbiota, and the obese microbiome seems to be more efficient in harvesting energy from the diet. Lean male donor fecal microbiota transplantation (FMT) in males with metabolic syndrome resulted in a significant improvement in insulin sensitivity and increased intestinal microbial diversity, including a distinct increase in butyrate-producing bacterial strains. Such differences in gut microbiota composition might function as early diagnostic markers for the development of T2D. The rapid development of FMTs provides hope for novel therapies in the future.

TriLink Products Cited in Microbiome Publications

It always amazes me to learn about the many ways TriLink products are used in basic and applied science. When I searched Google Scholar for publications containing “TriLink [and] microbiome” I found 21 items, among which the following were selected to illustrate diversity of these product types and uses:

Takeaway Messages

In summary, several takeaways should now be apparent to you. The first takeaway is that there is continuing explosive growth of microbiome publications in all manner of life-related research, as evidenced by both the introductory PubMed graph and wide spectrum of subjects covered by the Top 10 Cited Publications mentioned above.

The second takeaway is best summarized in publication #8 above, “[t]oday, animals and plants are no longer viewed as autonomous entities, but rather as ‘holobionts’, composed of the host plus all of its symbiotic microbes.” Each of us is indeed inextricably comprised of our human cells and symbiotic microbiota in or on us—like it or not, and for better or worse.

The final takeaway is that TriLink products play a contributing role in elucidating and applying this new world-view of halobionts.

As usual, your comments are welcomed.

Impossible Foods and Other Achievements of Pat O. Brown

  • Brown’s Microarray Publications Started a Revolution in DNA/RNA Analysis
  • Open Access Publishing Was an Unintended Consequence of His Microarray Research
  • Brown’s Passion for Bettering Earth Led to Invention of the Plant-Based Impossible Burger

Impossible Foods is a company founded by Patrick “Pat” O. Brown that wants to transform the global food system by inventing foods we love, without compromise. It’s first commercial product uses 0% meat and 100% plants to recreate everything—i.e. sights, sounds, aromas, textures and flavors—of a big, juicy burger, aptly named the Impossible Burger. “Impossible” because this was not thought to be doable, as many “veggie” burgers have fallen short on appearance, texture and—importantly—taste.

But before I tell you more about the circumstances and science of this game changer of a burger by Brown and company, let me start with his background as related to nucleic acid research, specifically microarrays.

Pat O. Brown and Microarrays

Pat O. Brown. Taken from Wikipedia.org

Brown received his BS, MD, and PhD degrees all from the University of Chicago, where he worked and published with Nicholas R, Cozzarelli on topoisomerases, which are enzymes that participate in the overwinding or underwinding of DNA. Brown did his postdoctoral research with uber-famous Nobel Laureates J. Michael Bishop and Harold Varmus at the University of California, San Francisco.

Brown went on to become a professor at Stanford University and in 1995 was the first to report (along with his colleagues) the use of microarrays for high-throughput analysis of nucleic acid. This seminal article published in venerable Science magazine and titled Quantitative monitoring of gene expression patterns with a complementary DNA microarray has now been cited more than 11,000 times in Google Scholar.

This publication described general methods for attaching cDNA probes for genes of interest onto glass microscope slides using a high-speed arraying machine (aka robotic printing). These were then hybridized to fluorescently labeled cDNA derived from mRNA by reverse transcription with dNTPs including labeled dCTP akin to dye labeled dNTPs offered by TriLink for such applications. Slides were then fluorescently scanned to obtain “spots” having pseudo-color intensities for quantitation relative to a “spike in” reference gene, as shown below.

Taken from Brown & coworkers Science (1995).

This paper triggered the genesis of what would become a highly competitive microarray industry, which I think of as going from “seeing spots to seeing dollars.” Interested readers can find much information about this in a review by pioneering experts during that time. A brief synopsis of this commercialization involving Brown is as follows.

From Microarrays to Open Access

Taken from plos.org

During his time at Stanford, Brown and his coworkers were using microarrays to generate huge amounts of data on gene expression profiling that required detailed analysis of even larger amounts of information previously published in many different journals. Although many of these journals were available online via a subscription, others were not, and almost all strictly forbade downloading and automated analysis. This thwarted Brown and others from compiling databases for anyone to use as needed. In other words, it prevented Open Access—allowing everyone, everywhere to have unrestricted, free access to this information.

Brown mulled over various ways for researchers to share their data, and in a coffee shop discussion with Harold Varmus, who was then Director of the NIH, they agreed on the possibility of a NIH-hosted computer server where scientists could post their work, and where it would be organized in a systematic way. Shortly thereafter in 1999, Varmus posted on the Director’s website a draft proposal for something that was dubbed e-Biomed.

In 2001, Brown helped lead the Public Library of Science (PLOS) initiative to make published scientific research open access and freely available to researchers in the scientific community. PLOS quickly grew in popularity, as have other Open Access journals, and PLOS now publishes roughly 20,000 papers per year. TriLink researchers—including yours truly—are pleased to be PLOS authors in a November 2016 report titled Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation, which has been viewed more than 2,700 times as of June 2017.

Impossible Foods

Taken from ajitvadakayil.blogspot.com

Apparently pioneering Open Access wasn’t enough for Brown and in 2009 he decided to devote his sabbatical to a daunting—if not impossible—challenge: eliminating conventional meat production from animals, which he estimated to be the world’s largest environmental problem, according to a reported interview. Reducing meat consumption, Brown reasoned, would free up vast amounts of land and water, as well as mitigate climate change due to methane emitted by animals (specifically, 8% of the world’s water and 15% of greenhouse gas emissions, according to one report). In addition, there would be elimination of enormous quantities of chemical fertilizers that are harmful to water systems.

Taken from impossiblefoods

‘All you have to do is make a product that the current consumers of meat and dairy prefer to what they’re getting now,’ Brown said and succeeded in raising $3 million in venture capital seed money. His startup company—aptly named Impossible Foods—then raised $108 million, in a whopper of a deal (pun intended) for development of its initial product—a plant-based burger, also aptly named the Impossible Burger.

The Impossible Burger is made from all-natural ingredients such as wheat, coconut oil and potatoes. What makes this burger unlike all other veggie burgers is an ingredient called heme. Heme is commonly associated with hemoglobin—the red pigment in blood—but is also found in other hemoproteins, including those in plants, albeit in low abundance compared to red meat. Therein lies the part of this story I decided to research: how does Impossible Foods obtain large amounts of plant heme having the desired properties for their burger?

Leghemoglobin taken from web.mst.edu

In an Impossible Foods patent by Brown et al., I found soy leghemoglobin identified as one such exemplary heme, which is pictured right. Plant cells within the nodule produce leghemoglobin to serve as an oxygen carrier to the bacteria within the nodule, similar to hemoglobin in blood. This enables the bacteria to obtain enough oxygen for respiration but ensures that the oxygen is in a bound form so that it cannot harm nitrogen fixing enzymes inside the bacteria. Cutting open a nodule reveals the red color typical of leghemoglobin when it binds oxygen, as seen below.

Impossible Foods biomanufacturing facility. Taken from psmag.com

According to the patent, biosynthetic leghemoglobin was expressed and purified using recombinant DNA technology for protein production, and then shown by SDS-PAGE gel and mass spectrometry to be identical to soybean leghemoglobin isoforms purified from soybean root nodules. Given the advanced state-of-the-art of industrial-scale recombinant production, I assume the Impossible Foods processes pictured right can be scaled-up to reduce cost.

If you think that Brown’s burger probably falls short of what you’d want for a meat substitute, think again. After watching an unbiased—I assume—and rather entertaining video of numerous taste testers (including meat eaters and a life-long vegan) give it positive reviews, I set out to sample an Impossible Burger. It was served as two sliders topped with sun-dried tomatoes, cavolo nero, vegan sun-dried tomato mayonnaise on a poppy seed bun served with chickpea panelle. I found the taste and texture nicely meat-like, but the $16 cost a bit tough to swallow—pun intended.

Meat-Substitutes are Ethically Compelling and Becoming Big Business

Readers interested in the ethically compelling case for developing meat substitutes like the Impossible Burger may be interested in a newspaper story about a for-credit curriculum now offered by the University of California, Berkeley. In that article, I was particularly impressed by the extent of other investments in commercialization of meat-substitutes:

  • In direct competition with Impossible Foods, which has raised a total of $183 million, Beyond Meat (which counts both multibillionaire Bill Gates and meats giant Tyson Foods as investors), sells The Beyond Burger™ as well as other meatless products.
  • In 2014, Pinnacle Foods (Vlasic® pickles, Birds Eye® vegetables) bought meatless food producer Gardein for $154 million.
  • Last year, Monde Nissin (instant noodles, etc.) of the Philippines purchased US-based Quorn for $831 million. Quorn’s fungus-derived mycoprotein can be processed to look and taste like chicken nuggets, sausage or patties.
  • Some startups such as Mosa Meat in Holland and Perfect Day in Berkeley are pushing the genetic engineering toward completely biosynthetic “meat” and “milk,” respectively, as recently reported in The Economist.

In conclusion, I think you’ll agree with me that the aforementioned accomplishments of Pat O. Brown give him good reasons to be smiling so broadly in his picture above. I certainly would be.

As usual, your comments are welcomed.

Postscript

After writing this blog, I read about Memphis Meats in San Leandro, California, which—like Mosa Meat and Perfect Day—has been developing cell culture-based technology to produce “meat.” Focusing on chicken, the company is quoted as saying that ‘the taste and texture is similar to that of the real thing, just a bit spongier.’ While this seems promising, it currently costs around $9,000 to produce a pound of Memphis Meats’ poultry, compared to a bit over $3 for a pound of chicken breast. However, the company hopes to reduce costs drastically and to launch a commercial product in 2021. I hope it does, but I think it won’t.

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Studying Telomeres in Space

  • Telomeres are DNA Biomarkers for Your Biological “Age”
  • Telomere Shortening Due to Stress was Expected During Spaceflight, but Exactly the Opposite has Been Found
  • Raising New Questions to Answer

After you read this blog about studying telomeres in space, I think you will agree with my opinion that scientific advances can sometimes occur amazingly fast. Telomeres (which are peculiar DNA structures that I’ll explain below) went from esoteric Nobel Prize subject matter in 2009 to the focus of spaceflight science in just six short years. Now, telomeres are being investigated by PCR on the International Space Station (ISS)! With a wink and a nod to Star Trek, this is indeed “warp speed” progress!

Taken from keywordsuggest.com

What are telomeres?

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates like us, the repetitive sequence of nucleotides in telomeres is TTAGGG, with the reverse complementary DNA strand being AATCCC, as depicted below. This sequence of TTAGGG is repeated ~2,500 times in humans.

During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, causing the end of the chromosome to be shortened in each replication. The telomeres are thus disposable “buffers” at the ends of chromosomes which are truncated during cell division, as depicted below.

Taken from weeklyglobalresearch.wordpress.com

Telomers, however, are replenished by an enzyme named telomerase. This peculiar enzyme has an embedded RNA template and incorporates DNA nucleotides, as depicted below, and is therefore a special kind of reverse transcriptase. In people, it has been found that telomeres shorten with age in all replicating somatic cells that have been examined. In fact, average telomere length declines from about 11 kilobases at birth to less than 4 kilobases in old age, with the average rate of decline being greater in men than in women. Thus, telomere length can serve as a biomarker of a cell’s biological (versus chronological) “age” or potential for further cell division.

Taken from 2014hs.igem.org

‘Houston, we have a problem’

This now famous phrase, which was used in the past tense by the crew of the Apollo 13 moon flight to report a major technical problem back to their Houston base, echoed in mind when I learned that space flight might lead to telomere shortening. Yikes! This molecular-level change could indeed be a serious problem, and was first suggested by findings from laboratory microgravity simulations reported in 2008 by Chinese researchers. Since it was known that space flight leads to bone loss, they cultured bone stem cells (BSCs) under simulated microgravity in a rotary cell culture system.

This led to significantly decreased activity of telomerase. It was postulated that reduced bone formation in space flight may partly be due to the altered potential differentiation of BSCs associated with telomerase activity, which plays a key role in regulating the lifespan of cell proliferation and differentiation. Additionally, telomerase activation or telomerase replacement may act as a potential countermeasure for microgravity-induced bone loss.

Taken from energeticnutrition.com

If you’re thinking that these “potential countermeasures” are fanciful, you’d better think again. I recently came across a patent that was published last year on methods and compositions for increasing telomerase activity in cells, including pharmaceutical formulations. Moreover, there are now various commercially available supplements claiming to promote telomerase activity, such as that picture below. I hasten to add that I do not advocate use of any such supplement, and that interested readers should consult their primary care physician or certified nutritionist.

Twins and telomeres

Although identical twins are almost the same genetically, differences in environment, diet and other outside factors can affect their health in different ways. Consequently, identical twins have been enrolled in various studies that require deciphering effects due to “nature vs. nurture” (i.e. intrinsic genetics vs. external factors). Part of the Twins Study supported by NASA was aimed at examining the effects of space travel on one of a pair of twins: astronaut Scott Kelly, who stayed on the ISS for one year, while his twin brother, Mark, remained on Earth. In brief, Prof. Susan Bailey at Colorado State University is exploring differences between the twins’ telomeres to determine if telomeres respond differently to spaceflight and then how such changes relate to the various medical endpoints studied by other Twins Study investigators.

Scott Kelly (left) and his identical twin brother Mark in 2015 prior to Scott’s one-year mission to the ISS. Taken from space.com

Preliminary research results for this part of the NASA Twins Study (reported at NASA’s annual Investigators’ Workshop earlier this year) were a quite surprising because they were opposite of what was expected, thus raising more questions than providing answers. It had been theorized that exposure to microgravity and stress during prolonged spaceflight would shorten telomeres, but instead Bailey’s team found telomeres in Scott’s white blood cells increased in length while in space! This finding was rationalized as being due to increased exercise and reduced caloric intake during the space mission. Upon his return to Earth, however, these telomeres began to shorten again.

This is yet another example of a biomedical phenomenon being far more complex that first theorized, and one that becomes less understood as more and more data are obtained. We’ll all have to patiently stay tuned for how this telomeres-in-space story evolves. The good news, as I’ll explore in the next and final section of this blog, is that there are exciting plans to use PCR to measure telomere length extraterrestrially! This is very “far out” science—pun intended.

Studying telomeres in space by PCR

Taken from geeky-gadgets.com

The aforementioned Twins Study involved taking blood samples from an astronaut during spaceflight for lab analysis upon return to Earth. To obtain much more data, and to do so in real time while in space, NASA launched the Genes in Space-2 mission in April 2017. The goal is to determine whether astronauts aboard the ISS can analyze telomeres by PCR reactions in a small thermal cycling device (miniPCR system) and thus measure and monitor telomere changes during spaceflight.

In addition to testing the miniPCR system, the Genes in Space-2 mission has a secondary goal to test the feasibility of techniques used to measure telomere length. Currently, Single Telomere Length Analysis (STELA) is the only suitable technique for use on the ISS due to technical requirements. The Genes in Space-2 mission will also be testing the feasibility of a loop-mediated isothermal amplification (LAMP) colorimetric assay for detection of amplification aboard the ISS. Please stay tuned for updates on the outcome of these very important feasibility experiments.

Scheme for STELA procedure. Taken from Xing et al. (2009)

Julian Rubenfein. Taken from nydailynews.com

As a side note, the Genes in Space competition for 2017 selected this experiment on telomere amplification in microgravity from 375 submissions by nearly 850 students in grades 7 to 12 from across the US. This telomere experiment was proposed by 17-year old Julian Rubinfien from Stuyvesant High School in New York City, who is pictured below. I encourage you to read this interesting, although lengthy interview about his background and the experimental rationale. What’s even more interesting is this short video of Julian at the launch and his comments—very impressive!

I strongly encourage you to read more about all the award-winning experiments in this exciting round of competition among young, highly motivated, advanced students, who I’m sure will be successful in whatever they do in the future.

Your thoughts or comments here are welcomed.

Postscript

Profs. Elizabeth Blackburn and Carol Greiner—who received a Nobel Prize in 2009 for seminal work on telomeres—co-founded Telome Health Inc. (THI) in 2010 to leverage the predictive power of telomere-length assays to help assess health status, disease and mortality risk, and response to specific therapies. THI subsequently announced TeloTest™ as a diagnostic test that measures average telomere length by qPCR. TeloTest™ was the first saliva-based telomere test available on the market, and is currently offered by a company named TeloYears.

The clinical utility of testing telomere length in a saliva-based test was recently reported from an independent, large clinical study sponsored jointly by Kaiser Permanente, University of California, San Francisco (UCSF), and National Institutes of Health. In the study, the average telomere length of 100,000 Kaiser patients was measured and analyzed relative to other health domains and clinical outcomes.

My recently obtained TeloTest™ results from TeloYears indicated that my biological age is 4 years older than my chronological age. Naturally, I was hoping to learn that my telomere-based age would be less than my actual age. Alas, the results are what they are, so I’ll be following diet, exercise, sleep, and stress-management recommendations you can read about at TeloYears Learning Center.

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CRISPR-C2c2 Update: Powerful New Diagnostic Method Using CRISPR

  • CRISPR Craze Continues Led in Part by Wunderkind Feng Zhang
  • Nonspecific CRISPR-C2c2 “Collateral” Cutting Channeled into Diagnostics
  • Turning Biochemical “Lemons to Lemonade”

This blog post is about a new and powerful diagnostic approach based on CRISPR, which I’ll get to below. But first I’d like to point out several reasons why this is an especially interesting development:

  • Taken from spyhollywood.com

    Any new method using CRISPR is more “sizzle” for this “super-hot” technology

  • Feng Zhang, the 32-year-old author of this publication, is regarded as a wunderkind
  • My past blog post on CRISPR-C2c2 “collateral” cutting noted possibilities for turning “lemons into lemonade”
  • Such “lemonade” has emerged as a diagnostic cleverly acronymized as SHERLOCK

So, without further ado, here’s a brief recap of CRISPR-C2c2 as an intro to SHERLOCK (think Holmes), followed by why this acronym is apropos for a diagnostic method that magnifies detection, in this case Zika virus, about which I’ve previously commented. 

What’s CRISPR-C2c2?

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems in microbes contain endonucleases that can be leveraged for CRISPR-based gene editing using targeted CRISPR RNAs (crRNAs), as I’ve outlined in a previous blog post. While such Cas enzymes target DNA, others target RNA and function as RNA-guided RNases, which was reported by Feng Zhang and collaborators in Science last year in an article titled C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.

My blog post about that C2c2 publication provides more information, but the main point related to the present blog post about diagnostics is that after recognition of its RNA target, activated C2c2 engages in nonspecific (aka “collateral”) cleavage of nearby non-targeted RNAs. This post-cleavage non-specificity can be transformed, if you will, from something “bad” to something “good” in terms of mechanisms, which I described as being akin to converting ‘lemons into lemonade’. You’ll now see how SHERLOCK is such ‘lemonade’ concocted by Zhang and coworkers.

What is SHERLOCK?

Taken from US 20140199245 A1

Before answering this question, and not to confuse you, I need to first mention that the nuclease originally called C2c2 was renamed Cas13a to be more systematic, and will be referred to herein as such. Having said that, Cas13a can be reprogrammed with crRNAs to provide a platform for specific RNA sensing. Molecular recognition of an intended RNA target by crRNA in vitro serves as a sequence-specific “trigger” to activate Cas13a’s nonspecific, “collateral” cleavage of labeled RNA reporters. Cleavage of these fluorescently labeled, internally quenched RNAs leads to unquenching and fluorescent light emission for detection, as depicted below for a generic RNase.

Now that you have a sense of how CRISPR-Cas13a can generate a signal, you’re probably asking ‘What is SHERLOCK?’ It’s an acronym coined by a large team lead by CRISPR genome-editing pioneer Feng Zhang and bioengineer James Collins, both from the Broad Institute. SHERLOCK stands for High-sensitivity Enzymatic Reporter UnLOCKing, which is a recently published new method for sequence-specific detection of DNA or RNA in any sample of interest. This general utility represents a major advance in CRISPR-based diagnostics.

Feng Zhang. Taken from be.mit.edu // James Collins. Taken from achetron.com

As depicted below, sample prep workflows allow for input of either double-stranded DNA (dsDNA) or RNA, which are first amplified by recombinase polymerase amplification (RPA) directly or as cDNA, after reverse transcription (RT-RPA), respectively. In either case, further amplification utilizes T7 transcription into RNA (blue = target RNA; yellow = non-target RNA). Following cleavage of target RNA by Cas13a-crRNA, a commercially available “cleavage reporter” (i.e. internally quenched) RNA undergoes “collateral” cleavage to generate a reporter signal (i.e. fluorescence; pictured below) for real-time detection of the target.

Taken from Feng Zhang, Collins & coworkers Science (2017)

An advantage of RPA is that it is performed isothermally, which allows simplification of the detection device by eliminating the need for a thermal cycler, thus lending to incorporation into small, portable point-of-care (POC) systems (about which I previously commented several times). The abovementioned RPA/T7 sample prep methodology was shown to have attomolar (aM, 10−18 mol/L) sensitivity in model systems, and was next investigated for sensitivity and specificity of virus detection, as well as simplification of reagent format.

Zhang, Collins & coworkers constructed lentiviruses harboring genome fragments of either Zika virus (ZIKV) or the related flavivirus Dengue (DENV), and showed that SHERLOCK detected viral particles down to 2 aM could discriminate between ZIKV and DENV. To explore the potential for POC use of SHERLOCK with paper-spotting and lyophilization (aka freeze-drying), they first demonstrated that Cas13a-crRNA complexes lyophilized and subsequently rehydrated could detect 20 femtomolar (fM, 10−15 mol/L) non-amplified RNA, and that target detection was also possible on glass fiber paper.

The other components of SHERLOCK, namely the RPA reagents and T7 polymerase, were already known to be amenable to freeze-drying and storage at ambient temperatures. In combination, freeze-drying and paper-spotting the Cas13a detection reaction resulted in comparable levels of sensitive detection of RNA as aqueous reactions. Although paper-spotting and lyophilization slightly reduced the absolute signal of the readout, SHERLOCK could readily detect mock ZIKV virus at concentrations as low as 20 aM. Most importantly, SHERLOCK detected ZIKV in clinical isolates (ZIKV RNA extracted from patient serum or urine samples and reverse transcribed into cDNA) could be detected at concentrations down to 1.25 × 103 copies/mL (2.1 aM), as verified by qPCR.

Concluding Comments

If you reflect upon the schematic for SHERLOCK, you’ll note that the input can be either DNA or RNA, which get amplified to produce many copies of RNA that serve as substrates for cleavage by Cas13a-crRNA, thus inducing collateral cleavage of reporter RNA to produce a detectable signal. Lest you think SHERLOCK is too costly to be practical, its developers provide a detailed cost accounting that estimates $0.61 per test, which you’re welcome to compare with your cost of conventional qPCR. I’m quite sure that will lead you to concur with me that $0.61 per test is relatively inexpensive.

If you’re questioning whether SHERLOCK is generally applicable, I urge you to read Zhang, Collins & coworkers in its entirety to learn more about SHERLOCK’s proven ability to detect and distinguish (1) various bacterial pathogens, (2) single-base cancer mutations in cell-free DNA, and (3) health-related single-nucleotide polymorphisms (SNPs) benchmarked against 23andMe genotyping data as the gold standard of these SNPs.

In an article in Science about SHERLOCK, Harvard University’s George Church (who I’ve proclaimed is The Most Interesting Scientist in the World and is the co-founded of a CRISPR therapeutics company), sums up his reaction in one word: ‘Wow.’ I agree!

The article concludes with Collins saying the Broad is now ‘aggressively exploring’ how to commercialize SHERLOCK and may launch a startup company. But before a diagnostic comes to market, it must pass muster at regulatory agencies such as the U.S. Food and Drug Administration. I’m betting is does.

I welcome your sharing any thoughts or comments about this new CRISPR-based diagnostic method.

Curiously Circular RNA (circRNA) Gets Curiouser

  • circRNA Molecules Have, Oddly, No Beginning or End
  • circRNA Are Now Recognized as Regulators of Gene Expression 
  • A Flurry of New Findings Indicate circRNA Are Also Templates for Synthesis of Proteins Having As Yet Unknown Functions

Electron micrograph of ~3,000-nt circRNA. Taken from Matsumoto et al. PNAS (1990).

About a year ago, my blog titled Curiously Circular RNA pointed out that circular RNA (circRNA) in animals are odd molecules in that, unlike the vast majority of other RNA in animals, circRNA have no structural beginning (5’) or end (3’). This very curious feature has, not surprisingly, stimulated considerable scientific interest in knowing more about these molecules, which were serendipitously discovered some 30 years ago.

Application of next-generation sequencing has revealed that circRNA are actually relatively abundant and evolutionarily conserved, which implicates biological importance rather than inconsequential mistakes during RNA splicing mechanisms. Some circRNA have been shown to have function—circRNA can hybridize to complementary microRNA (miRNA), and thus serve as a kind of ‘sponge’ that influences miRNA-based gene expression. Evidence for circRNA involvement in gene expression continues to grow, as there are now >700 items on “circRNA [and] sponges” in Google Scholar.

Very recently published lines of research (that I’ll outline in what follows) implicate circRNA as coding templates for proteins, which heretofore has been exclusively associated with messenger RNA (mRNA). Current dogma holds that translation of mRNA into protein requires recognition of the 7-methylguanylated (m7G) 5’-cap structure to start ribosome binding, while the 3’-poly(A) tail protects the mRNA molecule from enzymatic degradation and aids in stopping translation, as depicted below.

Taken from Shoemaker & Green Nature Structural & Molecular Biology (2012).

Start and stop structural elements characteristic of mRNA are obviously not present in circRNA, which are literally just circles of RNA. Consequently, finding proteins encoded by circRNA has stirred up controversy about whether such proteins are a new and fundamentally important aspect of genetics or just inconsequential biochemical mistakes.

Translation of circRNA in Fly Head Neurons

Fruit fly. Taken from turbosquid.com

Researchers at The Hebrew University of Jerusalem in Israel in collaboration with a team at Max-Delbruck-Center for Molecular Medicine in Berlin, Germany recently reported in Molecular Cell the first compelling evidence that a subset of circRNA is translated in vivo. The study by Kadener & coworkers was carried out using the common fruit fly (Drosophila melanogaster), which is known to have a number of features that lend to investigations of circRNA: (1) >2,500 fruit fly circular RNAs have been rigorously annotated, (2) these are mostly derive from back-splicing (pictured below) of protein-coding genes, (3) hundreds of which are conserved across multiple Drosophila species, and (4) exhibit commonalities to mammalian circRNA.

Direct back-splicing: a branch point in the 5’ intron attacks the splice donor of the 3’ intron. The 3’ splice donor then completes the back-splice by attacking the 5’ splice acceptor forming a circRNA. Taken from Jeck & Sharpless Nature Biotechnol (2014).

This study by Kadener & coworkers involves a plethora of technically complex experimental procedures and associated jargon, from which I’ve extracted what I believe to be some key points to share. After annotating the Drosophila circRNA open reading frames (cORFs), which, by definition,h have the potential for translation, they searched for evidence of their translation utilizing previously published ribosome footprinting (RFP). This led to identification of 37 circRNAs with at least one specific RFP read, referred to as ribo-circRNAs.

Taken from Jeck & Sharpless Nature Biotechnology (2014)

Several representative ribo-circRNAs were then constructed to each have (pictured below) a metallothionine (MT) promoter and V5 tag to facilitate translation and anti-V5 antibody-based detection of the expected protein after transfection into cells.

To determine whether circRNAs are translated in a more relevant tissue, they set up the RFP methodology in fly heads. A genetic locus named mbl that is known to produce a circRNA (circMbl3) at high abundance was selected for targeted mass spectrometry from a fly head immunoprecipitated MBL. They utilized synthetic peptides to determine characteristic spectra for which to search in the fly head immunoprecipitate and found a consistent and very high confidence hit for a peptide that can only be produced by circMbl3.

Kadener & coworkers extended these fly head findings to mammalian mouse and rat systems, but the most interesting part of this study—in my opinion—dealt with what signals ribosome binding and translation in the absence of the 5’ cap structure present in mRNA. They demonstrated circRNA translation under conditions intended to block normal 5’ cap-dependent translation of mRNA, and concluded that “[untranslated regions] of ribo-circRNAs (cUTRs) allow cap-independent translation [and that] further research is necessary to uncover how these sequences promote translation.”

Remarkably, as you’ll now read, another group of investigators have apparently found how such promotion of circRNA translation can occur.

Translation of circRNA is Driven by N6-Methyladenosine (m6A)

The most abundant modification of RNA in eukaryotes is m6A, which has been recently shown by Li et al. to recruit binding proteins that collectively facilitate the translation of specifically targeted mRNAs—i.e. those “marked” with m6A—through interactions with 40S and 60S ribosome subunit “machinery” that actually carry out translation. Contemporaneously, Yang et al. found that m6A likewise promotes efficient initiation of protein translation from circRNAs in human cells. They discovered that consensus m6A motifs are enriched in circRNAs, and a single m6A site is sufficient to drive translation initiation.

As depicted below, this m6A-driven translation requires initiation factor F4G2 and m6A “reader” YTHDF3. Experiments showed that this translation is enhanced by methyltransferase METTL3/14 and inhibited by demethylase FTO, which enzymatically “add” and “subtract” methyl (Me) groups on specific adenosines (A) in circRNAs, respectively.  It has also been shown to be upregulated upon heat shock, which is a commonly employed method to induce “stress” in cells.

Taken from Yang et al.

Further analyses through polysome profiling, computational prediction and mass spectrometry revealed that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Yang et al. concluded by stating that their “study expands the coding landscape of [the] human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.”

Zinc Finger Protein in Muscle Cell Development

Finally, and essentially contemporaneously with above mentioned two publications, a third independent investigation reported by Legnini et al. demonstrated selective circRNA downregulation using short-interfering RNAs (siRNAs). These reagents for RNA interference (RNAi) were used in an image-based functional genetic screen of 25 circRNA species, conserved between mouse and human, expression of which are differentially expressed during myogenesis (i.e. formation of muscular tissue) in Duchenne muscular dystrophy myoblasts.

This siRNA/RNAi-based functional analysis provided one interesting case related to zinc finger protein 609 (circ-ZNF609)—a reported miRNA sponge—the phenotype of which could be specifically attributed to the circular form and not to the linear mRNA counterpart. Consistent with the circ-ZNF609 sequence having an ORF, they found that a fraction of circ-ZNF609 RNA is loaded onto polysomes and that, upon puromycin treatment, it shifted to lighter fractions, similar to mRNAs. The coding ability of this circRNA was proved through use of artificial constructs expressing circular tagged transcripts, and by CRISPR/Cas9—the trendy gene editing method about which I’ve already commented multiple times.

Despite all this evidence, Legnini et al. stated that they “have no hints on the molecular activity of the proteins derived from circ-ZNF609 and as to whether they contribute to modulate or control the activity of the counterpart deriving from the linear mRNA.”

In thinking about closing comments about this update in circRNA, I decided to emphasize that investigations in the field of RNA continue to reveal complexities that will require many more years of global attention to unravel and understand. In just the past decade or so we’ve learned about gene regulation by miRNA/siRNA, reclassification of “junk DNA” as encoding a myriad of long noncoding RNA (lncRNA), mRNA regulation by base-modifications, and curious circRNAs that are more than sponges, and likely encode hundreds (if not thousands) of proteins whose functions have yet to be elucidated. Amazing!

What are your thoughts about all of this?

Your comments are welcomed.

Postscript

After writing this blog, Panda et al. at the National Institute on Aging-Intramural Research Program, National Institutes of Health published a paper titled High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Here’s a snippet of the abstract which adds to the increasingly curious occurrence of circRNAs that begs, if you will, further research aimed at discovering functions of circRNA-derived proteins.

“Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.”

National ALS Advocacy Day

  • Tuesday May 16, 2017 is National ALS Advocacy Day
  • Mark Your Calendar and Get Involved!
  • Read on to Find Out Why Involvement Matters

Lou Gehrig (1903-1941). Taken from Wikipedia.org

Amyotrophic lateral sclerosis (ALS) is more commonly referred to in North America as “Lou Gehrig disease.” Henry Louis “Lou” Gehrig was a record-setting baseball All-Star for the New York Yankees from 1923 through 1939, when he voluntarily took himself out of the lineup to stunned fans after his play was hampered by ALS. Sadly, Mr. Gehrig died only two years later, which indicates the rapidity of ALS disease progression.

ALS was first found in 1869 by French neurologist Jean-Martin Charcot, but it wasn’t until Lou Gehrig’s affliction that national and international attention was brought to the disease. ALS is now known to be a group of neurological diseases that mainly involve the degeneration of nerve cells (neurons) responsible for controlling voluntary muscle movement, like chewing, walking, breathing and talking. Motor neurons are nerve cells that extend from the brain to the spinal cord and to muscles throughout the body.

Taken from irelandms.com

The disease is relentlessly progressive, meaning the symptoms get continuously worse over time. Both the upper motor neurons in the brain and the lower motor neurons in the spine degenerate or die, and stop sending messages to the muscles. Unable to function, the muscles gradually weaken, start to twitch, and waste away (atrophy). Eventually, the brain loses its ability to initiate and control voluntary movements.

Currently, there is no cure for ALS and no effective treatment to halt, or reverse, the progression of the disease. Most people with ALS die from respiratory failure, usually within 3 to 5 years from when the symptoms first appear. However, about 10 percent of people with ALS survive for 10 or more years.

It is generally estimated there are over 30,000 people living with ALS in the United States at any given time, and that the number worldwide is around 450,000. Someone is diagnosed with ALS every 90 minutes. The life-time incident rate for an average person is often estimated at between 1-in-400 to 1-in-600 people—an incidence rate comparable to that for multiple sclerosis.

Who Gets ALS and Why?

Following are some reliable facts that I selected from an authoritative NIH website for ALS:

  • ALS affects people of all races and ethnic backgrounds.
  • Caucasians and non-Hispanics are most likely to develop the disease.
  • Although the disease can strike at any age, symptoms most commonly develop between the ages of 55 and 75.
  • Men are slightly more likely than women to develop ALS. However, the difference between men and women disappears with aging.
  • Military veterans are about 1.5 to 2 times more likely to develop ALS, and is recognized as a service-connected disease by the U.S. Department of Veterans Affairs.
  • 90% of ALS cases are considered sporadic, i.e. the disease seems to occur at random.
  • 10% of ALS cases are familial, i.e. an individual inherits the disease from his or her parents.
  • Mutations in more than a dozen genes have been found to cause familial ALS

How is ALS treated?

Riluzole. Taken from healthtap.com

No cure has yet been found for ALS. However, there are treatments available that can help control symptoms, prevent unnecessary complications, and make living with the disease easier. In 1995, the FDA approved riluzole (Rilutek), the only disease-modifying drug to date for ALS. Riluzole has multiple neural mechanisms of action, and is believed to reduce damage to motor neurons by decreasing levels of glutamate, which transports messages between nerve cells and motor neurons. Unfortunately, clinical trials in people with ALS showed that riluzole prolongs survival by only a few months.

BrainStorm Cell Therapeutics & NurOwn®

In researching clinical trials for hopefully much more effective therapies for ALS, I came across a company in Israel named BrainStorm Cell Therapeutics Inc. (BCTI) that offers a form of stem cell therapy for ALS that appears to be quite promising. Following is a short synopsis of what I found.

Space-filling model of BDNF. Taken from Wikipedia.org

BCLI has developed a patented stem cell-based technology that delivers a growth factor that can help neurons live longer at or near the site of injury or damage. More specifically, a mesenchymal stem cell isolated from an ALS patient is grown in a cell culture under certain conditions to produce a differentiated phenotype that secretes brain-derived neurotrophic factor (BDNF) at a level at least five-times greater than normal. The term “factor” is generic in biomedical parlance, and in the case of BDNF refers to a protein pictured to the right.

After these “super secreting” cells are obtained ex vivo, they are reintroduced (aka implanted) into the same ALS patient (i.e. an autologous transplant; see schematic diagram) wherein BDNF acts on neurons of the central nervous system and the peripheral nervous system, helping to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses.

Taken from Byrne JA. Overcoming clinical hurdles for autologous pluripotent stem cell-based therapies. OA Stem Cells 2013 Sep 01;1(1):3.

Details for this cell-harvesting, ex vivo differentiation, selection, and implantation can be read in this 2014 article by BCTI in Clinical Translation Medicine. BCTI has registered these differentiated BDNF-secreting cells as NurOwn®, which I assume is intended to sound bit like “your own”—to reflect the autologous nature of the transplant—and be a linguistic blend of neuron and own. In any case, the important point is that safety and efficacy have been reported in a recently published Phase 1/2 and 2a open-label, proof-of-concept studies of patients with ALS at the Hadassah Medical Center in Jerusalem, Israel.

In the Phase 1/2 part of the trial, 6 patients with early-stage ALS were injected intramuscularly (IM) and 6 patients with more advanced disease were transplanted intrathecally (IT), i.e. via the spinal cord. In the Phase 2a dose-escalating study, 14 patients with early-stage ALS received a combined IM and IT transplantation of autologous BDNF-secreting cells. Interested readers can consult this publication for details, but the bottom line, if you will, was that the possible clinical benefits would be hopefully confirmed in an upcoming clinical trial.

My further research on this led to a February 2017 press release by BCTI announcing that City of Hope’s Center for Biomedicine and Genetics (CBG) in Duarte, California will produce clinical supplies of NurOwn® adult stem cells for the BCTI’s planned randomized, double-blind, multi-dose Phase 3 clinical study in patients with ALS. It added that CBG is expected to support all U.S. medical centers that will be participating in the Phase 3 trial.

A second February 2017 press release by BCTI announced an agreement with Centre for Commercialization of Regenerative Medicine (CCRM) in Toronto, Canada to support the market authorization request for NurOwn® and explore the opportunity to access Health Canada’s early access pathway for treatment of patients with ALS as early as 2018.

Other ALS Clinical Studies

My “go to” authoritative source of reliable information about clinical trials is NIH’s ClinicalTrials.gov website that has an updated database that can be searched and filtered in many ways. When I searched for ALS clinical trials that were recruiting patients, I was heartened to find over 180 studies that can be perused via this link. For each study, there is information about purpose, study design and measures, eligibility, contacts and locations.

Incidentally, as regular readers of my blog will know, modified mRNA (mod mRNA) therapeutics is a relatively new and very promising modality for treating diseases that respond to providing or supplementing proteins. Given that DNA vectors encoding BDNF mRNA are readily available, I’m hoping that a mod mRNA for BDNF will soon be investigated as yet another avenue of treatment for ALS.

Advocate for ALS!

Taken from alsa.org

The ALS Association (ALSA) has the stated mission “to discover treatments and a cure for ALS, and to serve, advocate for, and empower people affected by ALS to live their lives to the fullest.” ALSA’s website offers various ways for any individual to become an advocate for ALS, such as becoming informed and donating much needed money or time, participating in the Walk to Defeat ALS® that draws people of all ages and athletic abilities together (see picture) to honor the courageous souls who are affected by ALS, to remember those who have passed, and to show support for the cause.

One of my long-time friends has recently been diagnosed with ALS, which in part led me to research this blog to help inform him, and led me to find a Walk to Defeat ALS® in which to participate. I encourage you to do advocate for ALS in whatever way you wish and are able.

As usual, your comments are welcomed.

DNA Day 2017

  • There are Now Millions of DNA-Related Publications
  • Some of the Top 5 Cited Papers on DNA Will Surprise You
  • You Probably Won’t Guess Top 5 Most Frequently Cited

Deciding what to post here in recognition of DNA Day 2017 was just as challenging as it has been in past years, primarily because there’s so many different perspectives from which to choose. After much mulling, and several abandoned approaches, I settled on featuring DNA publications that have received the most citations, as an objective metric—not just my subjective opinions about topics I think are significant or otherwise interesting.

Before getting to the numbers of DNA-related papers and some of the most cited papers, here’s a quick recap of what was posted here in the past, starting with the inaugural blog four years ago:

2013—60th Anniversary of the Discovery of DNA’s Double Helix Structure

2014—My Top 3 “Likes” for DNA Day

2015—Celebrating Click Chemistry in Honor of DNA Day

2016—DNA Dreams Do Come True!

Explosive Growth of DNA Publications

Regular readers of my blogs will know that I frequently use the NIH PubMed database of scientific articles to find publications by searching keywords, phrases, or authors. A convenient feature of these searches is providing “results per year” that can be exported into Excel for various purposes. Some preliminary searches indicated that DNA-related articles can be indexed by either DNA or PCR, or cloning, or other terms among which sequencing was notable. The majority, however, were indexed as either DNA or PCR, which together gave nearly 1.7 million items—an astounding number. This number is even much greater since PubMed excludes some important chemistry journals, as well as patents.

Diving deeper into these numbers, I thought it helpful to look at the publication volumes and rates for DNA, sequencing DNA, and PCR through 2015 starting from 1953, 1977, and 1986, respectively. These respective dates correspond to seminar publications by Watson & Crick, Maxam & Gilbert, and Mullis & coworkers. The results shown in the following graph attest to my often stated “power of PCR” as premier method in nucleic acid research, which we’ll see again below in another numerical context.

Top 5 Cited Papers

During my perusal of the above literature in PubMed generally related to DNA, I thought it would be interesting to find, and share here, which specific papers have the distinction of being most frequently cited. Citations are not available in PubMed, but are compiled in Google Scholar, which led me to these Top 5 that are listed from first to fifth.

Frederick Sanger (1918-2013) Taken from newscientist.com

  1. DNA sequencing with chain-terminating inhibitors

Frederick Sanger, the eponymous father of the “Sanger sequencing” method published in 1977, received the 1980 Nobel Prize in chemistry for this contribution. He also received the 1958 Nobel Prize in chemistry for sequencing insulin, and is the only person to win two Nobel Prizes in chemistry. Uber-famous DNA expert Craig Venter is quoted as saying that ‘Fred Sanger was one of the most important scientists of the 20th century,’ [who] ‘twice changed the direction of the scientific world.’

  1. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method

Kenneth J. Livak, PhD
Taken from archive.sciencewatch.com

The most commonly used method to analyze data from real-time, quantitative PCR (RT-qPCR) experiments is relative quantification, which relates the PCR signal of the transcript of interest to that of a control sample such as an

untreated control. The derivation, assumptions, and applications of this method were published in 2001 by Livak & Schmittgen. I overlapped with Ken Livak at Applied Biosystems, which pioneered commercilaization of RT-qPCR reagents and instrumentation at the time. He is currently Senior Scientific Fellow at Fluidigm Corp.

Sir Edwin M. Southern Taken from ogt.co.uk

3. Detection of specific sequences among DNA fragments separated by gel electrophoresis

Sir Edwin Mellor Southern, FRS, the eponymous father of “Southern blotting” DNA fragments from agarose gels to cellulose nitrate filters published in 1975, is a Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College. He is also Founder and Chief Scientific Advisor of Oxford Gene Technology.

  1. Prof. Bert Vogelstein, MD
    Taken from hhmi.org

    A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

This paper by Feinberg & Vogelstein published in 1983 describes how to conveniently radiolabel DNA restriction endonuclease fragments to high specific activity using the large fragment of DNA polymerase I and random oligonucleotides as primers. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments. His group pioneered the idea that somatic mutations represent uniquely specific biomarkers for cancer patients, leading to the first FDA-approved DNA mutation-based screening tests, and now “liquid biopsies” that evaluate blood samples to obtain information about underlying tumors and their responses to therapy (an area that I’ve touted in previous blogs). A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 109 dpm/μg of DNA can be obtained using relatively small amounts of precursor. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments.

  1. Kary B. Mullis, PHD
    Taken from TED.com

    Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

In 1988, Kary B. Mullis and coworkers (then at Cetus Corp.) published in venerable Science a method using oligonucleotide primers and thermostable DNA polymerase from Thermus aquaticus to amplify genomic DNA segments up to 2000 base pairs to detect a target DNA molecule present only once in a sample of 105 cells. Since that time, polymerase chain reaction (PCR)-related technology has evolved to now routinely enable a variety of single-cell analyses of DNA or RNA. Dr. Mullis received the 1993 Nobel Prize in chemistry for his 1983 invention of PCR, which his website says ‘is hailed as one of the monumental scientific techniques of the twentieth century.’

Top 5 Papers by Citation Frequency

While writing the above section, it occurred to me that ranking these five publications by total number of citations-to-date in Google Scholar doesn’t account for differences in the number of years between the year of publication and now. I did the math to calculate the average citation frequency per year, and here’s the totally surprising—to me—result: relative gene expression methodology published by Livak & Schmittgen is by far the most frequently cited of the Top 5, according to this way of ranking:

  1. 2001, relative gene expression, Cited by 69560 = 4,637 avg. citations per year
  2. 1977, Sanger sequencing, Cited by 32662 = 1,701
  3. 1975, Southern blotting, Cited by 21201 = 796
  4. 1988, PCR, Cited by 18785 = 671
  5. 1983, oligolabeled DNA, Cited by 21200 = 642

I should point out that, as transformative methods such as these gradually become widely recognized as “standard procedures,” researchers tend to feel it unnecessary to include a reference to the orignal publication. Consequenly, citation frequency decreases with time even though cummulative usage increases. In other words, 25 years from now average citations per year for relative gene expression will have likely decreased, and be surpassed by a new “method of the decade,” so to speak.

Prediction for the Future

This line of reasoning leads me to close with some speculation about what DNA-related technique might emerge as the next “method of the decade” that tops the above ranking by citation frequency.

My guess is that it will be Multiplex genome engineering using CRISPR/Cas systems by Zhang & coworkers that has been cited by 4145 at the time I’m writing this piece, only four years from its publication in venerable Science in 2013. Some of my blogs have already commented on various aspects of CRISPR/Cas9, which is among genome editing tools offered by TriLink.

As usual, your comments are welcomed.

Autism Awareness Month – April 2017

  • Sequencing for Diagnosis of Autism Holds Promise
  • Several Genetic-Risk Testing Procedures are Available
  • More Than 40 Autism Publications Using TriLink Products

The first National Autism Awareness Month was declared by the Autism Society in April 1970 with the aim of educating the public about autism. Autism is a complex mental condition and developmental disability, characterized by difficulties in the way a person communicates and interacts with other people. Autism can be present from birth or form during early childhood, typically within the first three years. Autism is a lifelong developmental disability with no single known cause.

The puzzle pattern of this ribbon reflects the complexity of autism, while the colors and shapes represent the diversity of people and families living with this spectrum of disorders. Taken from drdiane.com

People with autism are classed as having Autism Spectrum Disorder (ASD) and the terms autism and ASD are often used interchangeably. The term “spectrum” refers to the wide range of symptoms, skills, and levels of disability in functioning that can occur in people with ASD, which includes Asperger syndrome. Some children and adults with ASD are fully able to perform all activities of daily living while others require substantial support to perform basic activities. ASD occurs in every racial and ethnic group, and across all socioeconomic levels. However, boys are significantly more likely to develop ASD than girls. The latest analysis from the U. S. Centers for Disease Control and Prevention (CDC) estimates that 1 in 68 children has ASD.

Taken from myaspergerschild.com

According to the CDC, diagnosing ASD can be difficult, since there is no medical test, like a blood test, to diagnose the disorders. Doctors look at the child’s behavior and development to make a diagnosis.” More details from the CDC are provided at this link.

Notwithstanding this current difficulty for diagnosis of ASD, research has led to continuing progress toward possible blood tests for ASD, which is the focus of this blog and supplements an earlier posting here on treating autism with a broccoli nutraceutical.

ASD and Exome Sequencing

My Google Scholar search for “autism and sequencing” led to a mindboggling list of more than 47,000 items! When ordered by relevance rather than date of publication, two publications were each cited ~1,000-times following “back-to-back” appearance in venerable Nature magazine in 2012. This computes to a combined average of ~400 citations per year, or a fraction more than one citation per day on average, which to me signals significant attention by the ASD research community and thus worth commenting on herein.

Sanders et al., in the first of these two widely cited studies, carried out exome sequencing in 238 families wherein each pair of parents was unaffected by ASD but had a child who was affected (aka proband), and in 200 of these families there was an unaffected sibling. This study design feature is important in view of the widely held idea that complex personality traits are derived by a combination of “nature and nurture,” i.e. genetics inherited from parents and that which is learned or otherwise acquired by familial and all external events.

Before synopsizing what was found, I should note that germline single-base mutations spontaneously arise during mitosis in every generation, and are termed de novo single nucleotide variants (SNVs). Identifying SNVs remained refractory to analysis at the whole genome or exon level until the advent of next-generation sequencing (NGS) technologies.

Sanders et al. found that the total number of non-synonymous (i.e. changes in the amino acid sequence of proteins) de novo SNVs—particularly highly disruptive nonsense and splice-site de novo mutations—are associated with ASD. They concluded that their results “substantially clarify the genomic architecture of ASD, demonstrate significant association of three genes—SCN2A, KATNAL2 and CHD8—and predict that approximately 25–50 additional ASD-risk genes will be identified as sequencing [more] families is completed.”

Neale et al., in the second widely cited study, likewise conducted exome sequencing but on only 175 ASD probands and their parents. Nevertheless, they found that the proteins encoded by genes that harbored de novo non-synonymous or nonsense mutations showed a higher degree of connectivity among themselves and with previous ASD genes as indexed by protein-protein interaction screens. They concluded that their results “support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold,” but did acknowledge the strong evidence reported by Sanders et al. for individual genes as risk factors.

ASD Genetic-Risk Testing

The American Academy of Pediatrics (AAP) in 2013 issued a statement on ethical and policy issues for genetic screening of children for ASD that was prompted in part due to then recent progress by IntegraGen—a small French genomics company—on development of a gene test that uses a cheek swab to screen infants and toddlers for 65 genetic markers associated with autism. Highlights of the AAP’s statement include:

  • Genetic screening can be particularly useful for diagnosing older babies and children with developmental disorders such as autism.
  • Genetic screening should be made available for all newborns. However, parents should have the right to refuse screening after being informed of the benefits and risks.
  • The decision to offer testing or screening should be based primarily on the best interest of the child.

Taken from autismspeaks.org

By way of an update, I’m pleased to add that in a 2015 press release by IntegraGen it was announced that its ARISk® Test became the first test marketed in the U. S. to assess the risk of autism spectrum disorder in children. Among the following IntegraGen statements about the ARISk® Test, I think it’s most important to note the caveats I’ve bolded for emphasis:

  • The test does not confirm or rule out a diagnosis of ASD for the child tested.
  • The test is intended to be used together with a clinical evaluation and other developmental screening tools.
  • Intended for children with early signs of developmental delay or ASD and in children who have older siblings previously diagnosed with an autism spectrum disorder.
  • A genetic score, based on the total number of genetic markers associated with autism identified, is used to estimate the child’s risk of developing ASD.
  • Intended for use for children 48 months and younger. The ARISk® Test is not available for prenatal testing.

Taken from integragen.com

More recently, Courtagen—cofounded by my former Life Technologies colleague Kevin McKernan (coinventor of SOLiD® NGS)—has commercialized its sequencing analyses for ASD and other neurodevelopmental conditions. According to a Courtagen posting, “[i]n the absence of a known single-gene disorder, ASD likely involves a complex combination of both genetic and environmental factors that influence early brain development. Multi-gene panels, such as Courtagen’s devSEEK® panels, provide clinicians with information on a number of genes commonly associated with ASD and autistic features. Clinicians can then use information from multi-gene panels to tailor treatments that meet the patient’s unique genotype and symptoms.”

Some interesting—to me—logistical and operational information about devSEEK® (237 genes) is as follows:

  • Turn-around time for results is 4-6 weeks.
  • DNA for sequencing is extracted from a single saliva sample. No blood draw or muscle biopsy required; however, blood and muscle tissue are accepted.
  • Courtagen works with patients, physicians, and insurance carriers to pre-approve each test. Courtagen will bill the insurance company and is willing to handle an appeal process as needed.
  • A secure physician online portal is available for ordering genetic tests and accessing patient reports when completed. Genetic counselors are available to address questions regarding Courtagen test results.

ASD Research and TriLink

While mulling over how to conclude this Autism Awareness Month blog featuring genetic testing for ASD, I wondered about TriLink’s role in advancing autism research by virtue of its various nucleic acid-related products being used for autism investigations. I was pleased and proud to find more than 40 items by searching Google Scholar for articles with the words “autism and TriLink.”

Perusal of these items revealed that the most cited (450-times) report was a 2012 publication in highly regarded Cell titled MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System, which used TriLink 5-methyl-2′-deoxycytidine-5′-triphosphate (5m-dCTP). Given the apparent significance of this publication, I won’t try to give a short, simplified synopsis but rather quote the following part of the authors’ summary:

“We report that 5hmC [5-hydroxymethylcytosine] is enriched in active genes and that, surprisingly, strong depletion of 5mC [5-methylcytosine] is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.”

I also noted a 2016 Cutting-Edge Review in Arteriosclerosis, Thrombosis, and Vascular Biology titled A CRISPR Path to Engineering New Genetic Mouse Models. These investigators utilized TriLink Cas9 mRNA for gene editing analogous to that reported by others for CRISPR/Cas9-mediated knockout of the autism gene CHD8 (see above). This led to transcriptomic profiling showing that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.

In conclusion, I must say that I learned much new information about autism while researching this blog, which I hope you found informative as well as interesting. If so, I have achieved my goal of either increasing or reaffirming your awareness of autism, and the availability of genetic risk-assessment tests.

As usual, your comments here are welcomed.

Postscript

Recently, a team of academic researchers in Arizona made headlines with their publication in Microbiome reporting ties between autism symptoms and the composition and diversity of a person’s gut microbes, aka “gut microbiome,” about which I’ve commented on in several previous blogs.

The participants, who were 18 children with ASD (ages 7–16 years), underwent a 10-week treatment program involving antibiotics, a bowel cleanse, and daily fecal microbial transplants over 8 weeks. Remarkably, the new therapy seemed to provide some long-term benefits, including an 80% improvement of gastrointestinal symptoms associated with ASDs and roughly a 20% – 25% improvement in autism behaviors, including improved social skills and better sleep habits.

Click here for a simplified, educational video on this work by the principal investigator, Prof. James B. Adams at Arizona State University.

I should emphasize that this is a very small study, and much more research will be needed to verify and firmly establish possible benefits and risks. Interested readers should contact Prof. Adams regarding any questions they might have.