Small RNA is Big Science

  • Most Top-5 Citations in Clinical Chemistry are MicroRNA (miRNA) Biomarkers
  • miRNA Biomarker Bonanza is Predicted by Panel of Experts, Although No miRNA Biomarkers Have Yet Been Approved by FDA
  • Plethora of Potential Short Regulatory RNA Exists Beyond the Typical miRNA Microcosm

I’m always looking for new and hopefully engaging topics to comment on, and a recent “Best of Clinical Chemistry” item featured in a special issue of Clinical Chemistry definitely caught my attention. I wasn’t surprised by MIQE Guidelines being at the top, given that these are the “bible” for doing accurate quantitative PCR (qPCR) that has become a seemingly ubiquitous molecular assay for clinical studies. However, I was totally surprised that the next four “best of” all involved microRNA (miRNA)! Hence today’s blog about these small RNA being big science—play on words intended (although properly speaking I should say short rather than small).clinicalccIn view of this exciting Molecular Diagnostics Special Issue in January, find here the mostly highly cited Clinical Chemistry articles focused on molecular diagnostics for 5 years from 2009-1004.

  1. Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, and Carl T. WittwerThe MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments (Volume 55 Issue 4: April 2009)
  1. Jessica A. Weber, David H. Baxter, Shile Zhang, David Y. Huang, Kuo How Huang, Ming Jen Lee, David J. Galas, and Kai WangThe MicroRNA Spectrum in 12 Body Fluids (Volume 56 Issue 11: November 2010)
  1. Xu Ji, Rie Takahashi, Yumiko Hiura, Go Hirokawa, Yasue Fukushima, and Naoharu IwaiPlasma miR-208 as a Biomarker of Myocardial Injury (Volume 55 Issue 11: November 2009)
  1. Yi Zhang, Yin Jia, Ruiying Zheng, Yingjun Guo, Yue Wang, Hui Guo, Mingyu Fei, and Shuhan SunPlasma MicroRNA-122 as a Biomarker for Viral-, Alcohol-, and Chemical-Related Hepatic Diseases (Volume 56 Issue 12: December 2010)
  1. Taichi Adachi, Michio Nakanishi, Yoritaka Otsuka, Kunihiro Nishimura, Gou Hirokawa, Yoichi Goto, Hiroshi Nonogi, and Naoharu IwaiPlasma MicroRNA 499 as a Biomarker of Acute Myocardial Infarction (Volume 56 Issue 7: July 2010)

What Are miRNA?

An miRNA is a short (~22 nucleotides) non-coding RNA found in plants, animals, and some viruses that functions in RNA silencing and post-transcriptional regulation of gene expression. There are actually other functionally distinct short RNA, but I’ll defer getting into that “alphabet soup” by way of simplification until the end of this posting. Also by way of introduction, shown below is a self-explanatory cartoon of the basic miRNA pathway. Details for how miRNA function can be read in the links provided above, as the intent here is a brief segue to the aforementioned clinical applications, which frequently shorten—pun intended—miRNA to miR, and are generally hyphenated with a number up to 4 digits as an identifier.

The miRNA pathway. Taken from Chau & Brenner (2011).

The miRNA pathway. Taken from Chau & Brenner (2011).

High-throughput sequencing has enabled discovery of an increasing number of putative miRNA that can be found with related information in miRBase Blog that is regularly updated and currently lists a set of 1,996 “high confidence” miRNAs. Several years ago, TriLink BioTechnologies R&D team recognized the need for eliminating problematic “adapter dimers” during ligation of adapters to short RNA libraries prior to amplification and sequencing. This led to discovery of TriLink’s recently launched CleanTag™ Ligation Kit for Small RNA Library Preparation wherein chemically modified adapters greatly suppress adapter dimer formation without impairing ligation of short RNA.

I highly recommend reading a synopsis of the unlikely co-discovery of miRNAs first published in 1993 in back-to-back papers in prestigious Cell by Victor Ambros and Gary Ruvkuv, which goes back to their postdoctoral days at MIT. The significance of those seminal studies was recognized earlier this year by their each receiving $3M for the Breakthrough Prize.

Anbros and Ruvkun have many—dare I say 3M—reasons to smile. Taken from scientificamerican.com.

Anbros and Ruvkun have many—dare I say 3M—reasons to smile. Taken from scientificamerican.com.

Established in 2013, the Breakthrough Prizes are awarded in life sciences, physics and mathematics to recognize scientists and generate excitement about the pursuit of science as a career. Founded by technology and Internet entrepreneurs Sergey Brin and Anne Wojcicki, Jack Ma and Cathy Zhang, Yuri and Julia Milner, and Mark Zuckerberg and Priscilla Chan, the prizes are funded by grants from their foundations. Laureates are chosen by selection committees that comprise prior recipients of the prizes.

miRNA Biomarker Bonanza

Following these early discoveries, and coining the abbreviation miRNA in 2000, there have been over 41,000 publications indexed in PubMed, all of which are accessible from my PubMed search. These numbers show exponential yearly growth, with ~8,300 in 2014. That translates to an average of 23 miRNA related publications every day of the year—that’s a lot!

This massive amount of information has established that different sets of miRNAs are expressed in different cell types, tissues or fluids (see #2 on the list above), and has revealed multiple roles for miRNAs in animal and plant development. Importantly, aberrant expression of miRNAs has been implicated in numerous disease states (see #3-5 on the list above).

“Miner with Pick, Shovel and Pan” ca. 1850. Daguerreotype from the collection of Matthew R. Isenburg. Taken from georgetowndivide.wordpress.com

“Miner with Pick, Shovel and Pan” ca. 1850. Daguerreotype from the collection of Matthew R. Isenburg. Taken from georgetown divide.

Discovering correlations of one or more miRNA and/or other small RNA with diseases is now big science, so to speak, as well as potentially big business. These so-called “biomarkers” can be used as valuable molecular diagnostics—both clinically and commercially. Consequently, this rush—not unlike the Gold Rush of 1849—is optimistically characterized here as the “miRNA Biomarker Bonanza.”

Speaking of a bonanza—and pushing the metaphor further—I discovered a treasure trove of cached information about miRNA research and news at miRNA Blog. Interested readers should check it out for the latest updates and specific topics of interest. BTW, the blog also contains information on conferences, job/post-doc listings and if you’re hiring, job/post-doc candidates. Not surprisingly, there are also commercial adverts since that pays the bills.

MicroRNA Analysis: Is It Ready for Prime Time?

In 2012, Clinical Chemistry answered this rhetorical question by asking a series of leading questions to a panel of five eminent scientific experts. Rather than selectively paraphrasing these leading questions and, more importantly, each experts reply, interested readers can access the entire article here.

The key question—in my humble opinion—was the following: Is it premature to think that we could have US Food and Drug Administration (FDA)-approved kit-based assays for miRNA analysis in the near future? While all of the experts agreed that it was not premature, the following verbatim reply by Anna Szafranska-Schwarzbach—then Scientific Affairs manager at Asuragen—was the most informative:

“miRNAs will continue to be introduced into the clinical market as LDTs but will eventually result in FDA-approved kit products. The CLIA-based approach has many advantages, which include raising physicians’ awareness and increasing their comfort level with miRNAs as diagnostic tools. From a laboratory standpoint, an LDT allows refinement of the testing procedures based on improved understanding of the technical and clinical practicalities of miRNA testing before embarking on the path of kit development. However, the principles and procedures for diagnostic-kit manufacturing will likely become more widely adopted for the development of LDTs and the manufacture of test components and reagents. In the next 2 years, as the market matures and begins to adopt miRNAs as routine diagnostic analytes, miRNA-based LDTs should advance toward in vitro diagnostic-kit formats. This trend will likely be led by oncology-related products and, over time, extended to other clinical applications as well as companion diagnostics.”

FDA-Related Information on miRNA Biomarkers

Dr. Szafranska-Schwarzbach’s prediction got me interested in learning the FDA status of miRNA biomarkers. So, here’s what I found.

I revised my aforementioned search of miRNA on PubMed by adding AND diagnostics to the search criteria. The results whittled the initial 41,000+ publications down to 460 that interested readers can peruse here. After looking into some of these for a theme or other angle of interest, I decided to find miRNA biomarkers that have already proven valuable—as opposed to promising—by finding FDA-approved miRNA biomarkers for diagnostics.

That turned out to not be straightforward, but did lead to related information about the FDA’s position and active involvement with miRNA biomarkers that was all new to me, and worth sharing with you.

I initially found an FDA website that provides an up-to-date list (with links) entitled Nucleic Acid Based Tests. By definition, this list includes tests that analyze variations in the sequence, structure, or expression of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in order to diagnose disease or medical conditions, infection with an identifiable pathogen, and determine genetic carrier status.” While these are definitely worth knowing about, it’s unclear whether any of these involve miRNA.

On the other hand, searching the FDA website for miRNA biomarkers led to over 140 items that can be perused here. I’ve listed those that jumped out at me below. These drew my attention because they aren’t what I was expecting, i.e. sets of specific miRNA biomarkers for diagnosing specific diseases. Instead, they are sets of specific biomarkers for diagnosing toxicological and other problems, which to me was a clever twist and indicative of unmet medical needs.

Taken from harmondiscount.com

Taken from harmondiscount.com

Potential of Extracellular MicroRNAs as Biomarkers of Acetaminophen (APAP; aka Tylenol®) Toxicity in Children—this collaborative study by FDA and hospital researchers identified altered miRNA patterns in serum and urine from children as potential biomarkers of APAP toxicity. The data suggest that multiple serum miRNAs associate with liver injury, but the most notable appear to be miR-122 and miR-375 as they exhibited a high correlation with the level of serum APAP protein adducts. Urinary miR-940 and miR-375 were also elevated in subjects overdosed with APAP. In addition, the time course analyses provide a basis for future studies to validate miRNA as useful prognostic biomarkers in clinical setting.

Early Molecular Markers for Cardiac Tissue Injury Treated with Doxorubicin (DOXO)—Clinical utility of this this long-know, potent anticancer drug is limited by cardiotoxicity. A collaborative study by FDA and hospital investigators has identified 24 differentially expressed miRNAs following various doses of DOXO in mouse models. Importantly, certain miRNA changes were detectable before tissue injury occurred. To me this suggested monitoring “early warning” biomarkers to stop administering DOXO before cardiac damage takes place.

Taken from piximggif.com

Taken from piximggif.com

Evaluation of miRNA Signatures of Platalets During Storage—During storage under standard blood bank conditions, platelets normally start to lose their viability with time due to various changes collectively referred to as platalet storage lesion (PSL). Collaborating FDA and NIH researchers are studying stored platelets to find miRNAs that might can serve as biomarkers for determining the quality of stored platelets. Interesting, high levels of a single miRNA (miR-103b) may prove to be useful for monitoring PSL.

Close But No Cigar

While I strongly dislike the smell of cigar smoke, and don’t advocate smoking in general, I do like this old way of saying “to fall short of a successful outcome” and its interesting origin. But I digress, so back to the hope and promise for miRNA as diagnostic biomarkers, even though hope and promise sounds like current political pandering.

I decided to contact the FDA for an authoritative update as to whether any miRNA have been approved by the FDA as diagnostic or other biomarkers. So, I reached out to Dr. Qiang Shi, who co-authored the aforementioned study of miRNA as biomarkers of acetaminophen, and is with the FDA Innovative Safety & Technologies Branch, Division of Systems Biology, National Center for Toxicological Research in Jefferson, Arizona. The reply was that “[t]he FDA has not approved any miRNA based biomarkers.” Let’s hope that happens soon. 

Emerging Technologies and the FDA’s Acting Chief Scientist

Meet Dr. Luciana Borio here.

Meet Dr. Luciana Borio here >

In the aforementioned list of 142 miRNA biomarker items at the FDA website, I came across a massive (260+ page) pdf entitled 2015 FDA Science Forum: Emerging Technologies, May 27-28, 2015, which I plowed through to find the aforementioned snippets about DOXO and platelets. I was also pleasantly surprised to learn that the FDA now has a Chief Scientist, Luciana Borio, MD pictured right.

I say now, because when I worked for the FDA Center for Biologics Research Evaluation (CBER) at NIH there was no such position albeit 25+ years ago—yikes, time flies! On the other hand, I wasn’t surprised to read in her remarks at the Science Forum that “[m]any of the FDA’s 10,000 scientists are at work in [its] research laboratories…evaluating how emerging technologies are affecting FDA-regulated products. FDA then integrates the results of this research into [its] regulatory review process, enabling [its] reviewers to make science-based decisions about a [product’s benefits and risks.”

I say I’m not surprised by this statement because it’s an almost verbatim description of what we did at FDA/CBER “back in the day” when I was hired to learn about and establish automated DNA synthesis. At the time, the technique was a then emerging technology used by then emerging biotech companies seeking FDA approval using then emerging recombinant DNA technologies.

Technologies have matured and new ones—like miRNA biomarkers—have emerged, but not this important regulatory agency’s science policy, which is good thing.

Plethora of Potential Short Regulatory RNA Exists Beyond the miRNA Microcosm

As mentioned above, miRNA are just one subset of relatively short RNA (sRNA) that are known to exist and thought to largely play various roles in regulating gene expression. For reasons that will become obvious, interested readers will need to peruse the mentally dizzying details expounded in publications such as this recent review by Jha et al. Following is my short synopsis of the situation that has resulted from the advent of massively parallel deep-sequencing of RNA, which finds sequences per se, but not the function of these snippets.

Numerous sRNA have been associated with regulatory elements, but beyond that there is vagueness due to traditional approaches to identify miRNAs, limiting the otherwise vast world of sRNA mainly to hair-pin loop bred typical miRNA, according to Jhu et al. They therefore analyzed, for the first time, a huge volume of sequencing data from 4,997 individuals and 25 cancer types to report 11,234 potentially regulatory sRNA “which appear to have deep reaching impact.” Interestingly, the entire study is accessible as an interactive info-analysis portal handling more than 260 GB of processed data—that’s a lot! These researchers conclude that “[t]he possible degree of cell system regulation by sRNAs appears to be much higher than previously assumed.”

Stay tuned for more enlightenment of sRNA function as others jump into this hot trending field of nucleic acids research.

As usual, your comments are welcomed.

macro

Macrocosm and microcosm is an ancient Greek Neo-Platonic schema of seeing the same patterns reproduced in all levels of the cosmos, from the largest scale (macrocosm or universe-level) all the way down to the smallest scale (microcosm or sub-sub-atomic or even metaphysical-level). Taken from tumblr.com.

 

One thought on “Small RNA is Big Science

  1. I would be really interest in the data by Jhu et al. that you refer to. Could you please link the corresponding article or a PMID?

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