The 100,000 Genomes Project: What is it, What is its Status, and What Comes Next?

  • This Transformative Project was Announced by the UK Government in December 2012 
  • 100,000th Whole Genome was Sequenced Only Five Years Later
  • Rare Diseases and Cancers Continue to be the Focus
  • The European Union Seeks 1,000,000 Whole Genome Sequences by 2022

This past April 25th, DNA Day was recognized by scientists around the world to commemorate the day in 1953 when the double-stranded helical structure of DNA was proposed by Watson & Crick in their famous publication in Nature titled Genetic Implications of the Structure of Deoxyribonucleic Acid. During the preparation of my blog for DNA Day 2019, I read The Double Helix: A Personal Account of the Discovery of the Structure of DNA by Watson, which I highly recommend, especially to anyone who works with DNA. I also read DNA: The Story of the Genetic Revolution, also by Watson, who provides an excellent educational narrative of how elucidation of DNA has impacted basic and applied health-related sciences.

This blog on the 100,000 Genomes Project was inspired by the latter book’s chapters on how the evolution of faster, better, and cheaper genomic sequencing has led to “The Age of Personalized Medicine,” which has been defined as follows:

Personalized medicine is the tailoring of medical treatment to the individual characteristics of each patient. The approach relies on scientific breakthroughs in our understanding of how a person’s unique molecular and genetic profile makes them susceptible to certain diseases. This same research is increasing our ability to predict which medical treatments will be safe and effective for each patient, and which of these will not be.

Taken from commons.wikimedia.org and free to use.

Continuing improvements in “faster, better, cheaper” DNA sequencing methodologies are essential to personalized medicine. In striking contrast to the estimated cost of between $500 million to $1 billion over 13 years to sequence the first human genome, which was completed in 2003, massively parallelized (aka high-throughput) genome sequencing can now be achieved in factory-like facilities for an estimated cost of around $1,000 per genome, as pictured here. 

In regard to low cost, Dante Labs—an Italian startup company— announced its launch of the first commercial long-read whole genome sequencing (WGS), with 30X coverage, for $999 using Oxford Nanopore technology, in April 2019. In regard to speed, a new ultra-fast, ultra-high-throughput sequencer from MGI Tech Co., Ltd. in China can complete WGS for up to 60 human genomes in a single day, according to a recent news article. For more information about advances in DNA sequencing, interested readers may refer to a review article published in 2017 by Shendure et al. titled DNA sequencing at 40: past, present and future.

What Is the 100,000 Genomes Project?

In 2012, the UK government announced the establishment of the 100,000 Genomes Project, which would revolutionize patient diagnosis and treatment by offering the prospect of personalized treatment to many patients. The project is run by Genomics England, a company that is wholly owned by the Department of Health, but is delivered through 13 Genomic Medicine Centers.

The key goals of the project are to promote genetic research in order to benefit patients, and to support the development of the UK genomics industry. WGS is carried out by a single provider, Illumina, at a purpose-built facility in Cambridgeshire. The program currently has two medical targets: rare diseases and cancer. It is designed as “a transformative project and to make interpretation of our DNA sequence sit alongside conventional diagnostic procedures and tests to inform the appropriate clinical pathways for treatment of disease,” according to an article in The Biomedical Scientist by Gerry Thomas, Professor of Molecular Pathology.

Thomas added that, for rare diseases, only a blood sample is required—usually from the patient and his or her parents. Most of these families are already aware that an inherited genetic component to their disease is a likely cause. For cancer patients, a paired sample of blood and tissue is required, the latter of which posed technical issues that are discussed in the next section.

What is the Status of the 100,000 Genomes Project?

The feasibility of WGS for cancer patients in the clinic has focused on the use of high-quality nucleic acids extracted from fresh-frozen tissue (FF) specimens collected within a research infrastructure. However, FF specimens are not routinely collected, as formalin-fixed, paraffin-embedded (FFPE) material is the specimen of choice for histopathological diagnosis, as pictured here. DNA extracted from FFPE specimens is somewhat degraded due to fragmentation, DNA crosslinks, abasic sites, and deamination leading to C>T mutation artifacts, which impede downstream sequencing analysis.

Robbe et al. addressed these technical issues in a pilot feasibility study that evaluated WGS data sets obtained from 156 genomes from 52 matched FF tumor, FFPE tumor, and peripheral blood samples routinely collected as part of the diagnostic process. The differences observed between FF and FFPE sequence data allowed for the development of new methods to optimize the quality of FFPE-derived WGS data, and allow the acquisition of genome-wide data for all patients with cancer, including those for whom only FFPE material is available.

I find it quite remarkable that  on December 5, 2018, after only six years since its conception, Genomics England announced that the Project had reached its goal of sequencing 100,000 whole genomes from patients. More importantly, this effort already realized the following benefits:

“The 100,000 Genomes Project has delivered life-changing results for patients, with one in four participants with rare diseases receiving a diagnosis for the first time, and providing potential actionable findings in up to half of cancer patients where there is an opportunity to take part in a clinical trial or to receive a targeted therapy.” 

Among the many video-stories shared by patients and their families available on the Genomics England website, I selected an example of a rare disease and a cancer as representative of these “life-changing results” delivered from the 1000,000 Genomes Project.

Jessica’s Story: Jessica, aged 4, was afflicted with a rare condition of unknown cause. She and her parents gave a small sample of blood and their genomes were sequenced. Every genome is compared to the reference human genome sequence, which is used as a guide, and Jessica’s genome had 6.4 million single-nucleotide differences (aka variants). Because Jessica’s condition was rare, and not shared with either parent, the next bioinformatic step looked for rare variants not present in the genome of either parent, but predicted to cause a change in an encoded protein. Out of 67 such variants, only one was located in a gene listed in PanelApp as being linked to symptoms similar to the ones Jessica was experiencing. To me, this is the equivalent of systematically finding a “genetic needle in a genomic haystack”!

The name of the gene in question is SLC2A1, and in Jessica’s genome, a single frameshift-mutation prevents expression of the encoded protein from that copy of the gene, meaning that she doesn’t have enough in her body. The SLC2A1 gene makes the glucose transporter protein type 1 (GLUT1), which is involved in moving glucose, the brain’s main energy source, across the blood-brain barrier. Two normal copies of this gene are needed for this protein to transport enough glucose to fuel the brain. Mistakes in the SLC2A1 gene can cause ‘GLUT1 deficiency syndrome’, which is Jessica’s diagnosis. Research has shown that in some patients who have GLUT1 deficiency syndrome, a special low-carbohydrate (ketogenic) diet can help reduce the number of seizures they experience by providing an alternative energy source for the brain.

The Lloyd Sisters’ Story: Three sisters, Mary, Sandra, and Kerry Lloyd, all developed breast cancer within 15 months of each other. Their late mother had also been affected, as well as two other female relatives over three generations. They had already undergone genetic testing for changes in the BRCA1 and BRCA2 genes, which can be a cause of breast cancer, but these tests were negative. The sisters each gave a small amount of blood to have had their genomes sequenced so that bioinformaticians would be able to analyze the sequences and determine the nature of their breast cancers.

BRCA1 tumor suppressor protein RING domain, in complex with BARD1 protein, as discussed elsewhere.

Breast cancer affects approximately 1 in 8 women in the UK, and about 5-10% of breast cancers are thought to be hereditary, caused by abnormal genes passed from parent to child. This phenomenon is known as ‘familial breast cancer’. Families that have familial breast cancer may include men with breast cancer, and are sometimes afflicted by other cancers as well, such as ovarian cancer or prostate cancer. In these families, cancers may develop at a younger age than usual. Most inherited cases of breast cancer are associated with specific changes in BRCA1 and BRCA2, genes that are present in females and males. 

The function of the BRCA genes is to repair cell damage and keep breast cells growing normally. When these genes contain abnormalities or mutations, they don’t function normally, and breast cancer risk increases. However, not all familial breast cancer is due to mutations in BRCA. Changes in other genes have also been found to play a role in causing familial breast cancer. By taking part in the 100,000 Genomes Project, the Lloyd sisters are contributing to research. Their de-identified data, together with data from other participants, is available to researchers through a secure database. There is great interest in understanding the genomic factors connected to breast cancer risk, which could include changes in the way known risk genes (like BRCA1 and BRCA2) are regulated, or switched on and off.

What Is Next for the 100,000 Genomes Project?

On March 4, 2019, Genomics England’s Chief Scientist and interim Chief Executive Professor Mark Caulfield reported on what is next for 100,000 Genomes Project participants:

  • The first priority is to get reports back to those who have not yet received a result.
  • The second priority is to revisit the genomes for people who have not yet gotten an answer, to see if new knowledge or new ways to analyze a genome will be able to find answers.
  • Panels of genes based on worldwide literature will be expanded to include recently added information, and other parts of the genome. The virtue of having a person’s whole genome is the ability to reanalyze it with new knowledge to get new answers for the participants.
  • The current goal is to analyze the genome of all participants for the first time, and return the results by July 2019. Scientists and doctors will then study the reports and consider whether there is adequate reliability to give the information back to the participants, as the clinicians on the receiving end are those caring for patients.

Other forward-looking information I found on the Genomics England website and thought worth mentioning, is related to various partnerships exemplified by the following: 

  • Genomics England signed a strategic research and development agreement with the Qatar Genome Program. This includes standardization of genomic strategies for healthcare implementation; the evaluation of new technologies for WGS; the cross-analysis of both national datasets; and the exchange of expertise related to educational programs.
  • American pharmaceutical companies Alexion and BioMarin, both members of Genomics England’s Discovery Forum, have identified previously undiagnosed patients with rare life-threatening kidney and neurological diseases, respectively. Nephronophthisis (NPHP) is responsible for 15% of cases of childhood end-stage renal failure. Neuronal ceroid lipofuscinoses 2 (CLN2) (aka Batten Disease) symptoms typically emerge in children aged 2-4, who have a life expectancy of around 10 years. 
  • Genomics England announced the successful completion of the first phase of its collaborations with Inivata in the UK and Thermo Fisher Scientific in the US to investigate the use of liquid biopsies in cancer, which I have previously blogged about. This is part of a pilot project to assess the suitability of circulating tumor DNA (ctDNA) samples. The results of the study showed that 200 plasma samples from the 100,000 Genomes Project across all cancer types were of a high quality and produced reliable results.

1,000,000+ Genomes Initiative by the European Union 

If you think that the 100,000 Genomes Project by the UK is impressive, then the European Union’s (EU) plan to obtain 1,000,000+ human genome sequences by 2022 will strike you as amazing. Here’s a brief synopsis of this initiative, according to statements on the European Commission policy website. 

Since its launch on Digital Day 2018, the “1+ Million Genomes” initiative has grown into a cooperative effort involving all 20 signatory Member States and Norway. These countries meet on a regular basis in order to ensure that the aim of the declaration—having at least 1 million sequenced genomes available in the EU by 2022—is achieved. This includes linking access to existing and future genomic databases across the EU, as well as providing a sufficient scale for new clinically impactful associations in research. The expected benefits to EU citizens are summarized as follows:

“Genomics has the potential to revolutionize healthcare in many ways. It could lead to the development of more targeted personalized medicines, therapies and interventions. It could also enable better diagnostics, boost prevention and make more efficient use of scarce resources. From cancer, to rear diseases, brain related diseases or prevention—Genomics can greatly improve various health conditions of EU citizens. Equally important, Genomics has also the potential to improve the effectiveness, accessibility, sustainability and resilience of health systems in the EU.”

Closing Comments

It is important to note that all participants in the 100,000 Genomes Project are patients of the UK National Health Service (NHS), which is the publicly funded national healthcare system for England, and one of the four National Health Services of the UK. It is the largest single-payer healthcare system in the world. Primarily funded through the government and overseen by the Department of Health and Social Care, NHS England provides healthcare to all legal English residents, with most services free at the point of use. 

As usual, your comments are welcomed.

Addendum

After this blog was written, there was a report that California state legislators have introduced a bill to increase WGS access to pediatric illnesses. The bill is referred to as “Ending the Diagnostic Odyssey Act.” The aim of the bill is to provide access to WGS for “certain undiagnosed children under the Medicaid program, and for other purposes.”

The bill is supported by Rady Children’s Hospital in San Diego and is also sponsored by Rep. Juan Vargas (D-CA). The belief is that rapid WGS should be available at hospitals nationwide for critically ill infants and children. Rady Children’s Hospital-San Diego has pioneered use of rapid sequencing in the care of pediatric patients.

Surge in Lyme Disease Cases Reported to U.S. Congress as a Serious Health Concern

  • Tick-Borne Diseases Working Group Reports Concern to U.S. Congress
  • Lyme Disease is the Major Tick-Borne Disease of Concern
  • 30,000 Lyme Disease Cases Confirmed, But 300,000 Cases Likely
  • Improved Vaccine Development and Public Relations Are Recommended 

A lengthy 2018 report to the U.S. Congress by a Working Group of experts has warned that “diseases transmitted by ticks are a serious and growing public health concern.” The report adds that 20 medical conditions can result from tick bites, with a 21st recently identified. Lyme disease (LD) is by far the most common condition. Over the past 25 years, cases of LD have increased steadily by 300%, with 30,000 new cases confirmed in 2018, and 300,000 cases estimated in the U.S.

Tick with its chelicerae (pincer-like mouth parts) sticking in human skin.

Interested readers can consult this 2018 report for details on the various tick-borne diseases (TBD) and, importantly, all of the recommendations made to Congress. Selections from these recommendations will be briefly reported on in this blog, together with background on LD, DNA-based tests for LD, and vaccines for prevention of LD: past, present, and future—the latter of which includes the possible use of modified mRNA vaccines.

Background on LD

Tick bite “bull’s- eye” pattern.

As detailed elsewhere by Steere et al., LD was so-named in 1976 because of the geographical clustering of children in the area of Lyme, Connecticut, who were thought to have juvenile rheumatoid arthritis. It then became apparent that Lyme arthritis was a late manifestation of LD associated with an insect bite. Many people with early-stage LD develop a distinctive circular rash (erythema migrans, EM) at the site of the bite that is described as looking like a “bull’s- eye” on a dart board, as seen here. Other symptoms include fever, chills, fatigue, headache, numbness in hands and legs, joint pain, and short-term memory loss. Antibiotics are used to treat the infection. 

In 1981, Burgdorfer and colleagues discovered a previously unidentified spiral-shaped bacterium, named Borrelia burgdorferi. (B. burgdorferi), in a black-legged tick (Ixodes scapularis; aka deer tick), both of which are pictured here. The bacterium was cultured from patients with early LD, and patients’ immune responses were linked conclusively with the organism. Based on genotyping of isolates from ticks, animals, and humans, B. burgdorferi has now been subdivided into multiple Borrelia species, including three that cause human infection. The sole cause of LD in the U.S. is B. burgdorferi. Although all three species are found in Europe, most disease there is caused by B. afzelii or B. garinii, and only these two species seem to be responsible for the illness in Asia.

3D illustration of B. burgdorferi.

Close-up photo of a deer tick on piece of straw.

According to Steere et al., LD continues to flourish and spread because of evolving climatic conditions that are especially favorable for B. burgdorferi infection between animals (enzootic). This is explained and schematically depicted elsewhere by the researchers. While originally associated with favorable weather in northeastern U.S., a recently published study found B. burgdorferi in a variety of mammalian and avian hosts throughout Canada. These findings further demonstrate the ability of this pathogen to spread across wide geographical areas, and investigators warn of “a serious public health contagion Canada-wide.” Such findings may equally apply to other parts of the world.  

The Borrelia Genome and Pathogenesis

Circular plasmid illustration.

Before commenting on DNA-based tests for Borrelia in the next section, I think it’s important to discuss this pathogen’s unusual genome. The determination and analysis of the first Borrelia genome sequence, B. burgdorferi type strain B31, stimulated significant progress in the biology, genetics and molecular pathogenesis of Borrelia, according to Casjens et al. Its unusual genome is comprised of a 910 Kbp linear chromosome and twenty-one (twelve linear and nine circular) plasmids that contain over 600 Kbp of DNA (two additional plasmids are now thought to have been lost between the isolation of strain B31 and its genome sequence determination). Casjens et al. further note that many other studies have shown that Borrelia isolates universally harbor numerous linear and circular plasmids. The B31 chromosome carries 815 predicted genes that largely encode for housekeeping functions. These functions include minimal metabolic capabilities, meaning that the pathogen cannot synthesize amino acids, nucleotides, or lipids de novo.

The pathogenesis of B. burgdorferi is very complex and beyond the scope of this blog. Some key points worth noting are that this pathogen is highly invasive, but does not produce any toxins. LD pathology is generally thought to be the result of host inflammatory response. Interested readers can consult a review by Petzke and Schwartz for details. 

Nucleic Acid-Based Tests for LD

96-well plate-based qPCR.

According to Eshoo et al., early diagnosis of LD and correct treatment generally lead to excellent outcomes. Direct Borrelia molecular tests such as PCR from blood can detect and identify active infection sooner than serologic tests, but have suffered from low assay sensitivity for clinical use. For example, early studies using PCR to detect Borrelia in the blood during active infection had limited success, displaying sensitivities of only 18%-26.1%. In recent years, studies have reported a higher detection rate through sampling of larger blood volumes (down to 10 genome copies), by culturing prior to PCR, and by using different PCR techniques such as qPCR or nested PCR.

To investigate further improvements, Eshoo et al. have studied detection of Borrelia DNA using previously commercialized PCR in combination with electrospray ionization mass spectrometry (PCR/ESI-MS). The basis of this detection and identification method is a multi-locus broad-range PCR, followed by determination of the mass of amplicons using automated ESI-MS. From the masses of amplicons, the numbers of DNA base pairs in each amplicon are determined. By analysis of the base compositions of amplicons from all primer pairs, the organisms present in the sample can be identified and quantified through a database of all known base count signatures. This technique has the advantage of rapidly identifying and genotyping pathogens, as well as identifying new genetic variants. The results demonstrated improved sensitivity using 1.25 mL of whole blood and an isothermal amplification for Borrelia DNA on the entire specimen extract prior to a multi-locus PCR/ESI-MS assay.

I researched high-throughput (aka next-generation) sequencing (NGS) for LD diagnosis, and found a recently initiated clinical trial that is investigating NGS for detection of B. burgdorferi DNA in blood of pediatric patients with Lyme-related EM rash. This pilot study will compare 20 study participants with the EM rash (cases) to 10 healthy study participants without an EM rash (controls). The investigators will compare currently used Lyme testing and NGS in both the cases and controls. The case participants will then have two follow-up appointments at 1-3 weeks and 2-3 months later, to obtain updated clinical information and repeat NGS testing. Basic descriptive statistics on the results will be used to determine if NGS is capable of detecting Lyme DNA during the acute phase of infection.

It’s also worth mentioning that Dr. Robert Moritz at the Institute for Systems Biology recently received research funding totaling nearly $525,000 from the NIH to develop novel peptide-based biomarkers for Lyme disease diagnostics. The Moritz group has been at the forefront of the development of whole-proteome assay repositories and will apply this technology to B. burgdorferi. 

Vaccines for LD: Past, Present, and Future

Dr. Stanley A. Plotkin. Taken from commons.wikimedia.org and free to use.

The vexing history of a vaccine against LD is perhaps best summarized by Dr. Stanley A. Plotkin, pictured below. He is widely acknowledged as an expert in vaccine development, for which he has received numerous awards. His publication titled Correcting a Public Health Fiasco: The Need for a New Vaccine against Lyme Disease states that “[a] vaccine against LD was licensed in the U.S. in 1998 but was subsequently removed from the market because of lack of sales. [He believes] that the poor acceptance of the vaccine was based on tepid recommendations by the Centers for Disease Control and Prevention (CDC), undocumented and probably nonexistent safety issues, and insufficient education of physicians.” 

A YouTube presentation by Dr. Plotkin and other experts on this former Lymerix™ vaccine “fiasco” also discusses the present status of vaccine development, which is well worth listening to, in my opinion. Alternatively, or in addition, there is a detailed review article by Embers and Narasimhan titled Vaccination against Lyme disease: past, present, and future.    

The TBD Working Group mentioned in the introduction was established by Congress in 2016 as part of the 21st Century Cures Act. It reviews federal efforts related to all TBDs, in order to help ensure interagency coordination and examine research priorities. The TBD Working Group creates a report on its findings, and provides recommendations regarding the federal response to TBD prevention, treatment, research, as well as the notable gaps in these areas.  

The 2018 report, which is 108 pages, can be accessed as a PDF at this link.  Among its recommendations, the following were favorably endorsed by LymeDisease.org, a non-profit group that serves the LD patient community through advocacy, education, and research:

• Build trust via a transparent mechanism by which all stakeholders examine and discuss past vaccine activities and potential adverse events to inform future vaccine development in Lyme disease.

• Support the development of safe and effective human vaccines to prevent Lyme disease with transparent mechanisms by which all stakeholders examine and discuss past vaccine activities and potential adverse events to inform future vaccine development.

• Prioritize education by informing clinicians and the general public about the regional and specific risks related to tick-borne diseases.

I’m optimistic that the priorities set by Congress for the National Institutes of Health and CDC, alongside the recent infusion of federal funding, will likely lead to an improved vaccine in the near future. However, I believe the hardest part will be the widespread adoption of such a vaccine in light of the ongoing vaccine resistance (‘anti-vax’) movement, which I have recently blogged about. In a recent publication that characterizes the anti-vax phenomenon as a “regression in modern medicine,” Hussain et al. describe some of the reasons behind the recent strengthening of the movement, the role of the internet in the spread of anti-vaccination ideas, and the repercussions in terms of public health and safety.  

To end on a positive note, I searched Google Scholar for Lyme and TriLink and found 33 items, the majority of which are patents that use modified mRNAs as potential vaccines against LD. This experimental interest in possible mRNA vaccines for safe and effective protection from LD is consistent with the current interest in modified mRNA, which I have previously blogged about. Readers interested in modified mRNA can find brochures, posters, and videos by clicking TriLink Education. 

As usual, your comments are welcomed.

Addendum

After I wrote this blog, the CDC issued a press release on the disease threat posed by the fast-multiplying Asian long-horned tick, which is quickly spreading across the U.S. and looks similar to the tick shown here. “The full public health and agricultural impact of this tick discovery and spread is unknown,” said Ben Beard, PhD, deputy director of CDC’s Division of Vector-Borne Diseases.  “In other parts of the world, the Asian long-horned tick can transmit many types of pathogens common in the United States. We are concerned that this tick, which can cause massive infestations on animals, on people, and in the environment, is spreading in the United States.”

The press release adds that New Jersey was the first state to report the tick on a sheep in August 2017. Since then, 45 counties or county equivalents in New Jersey and eight other states—Arkansas, Connecticut, Maryland, North Carolina, New York, Pennsylvania, Virginia, and West Virginia—have reported finding the tick on a variety of hosts, including people, wildlife, domestic animals, and in environmental samples. On May 31, 2019, Wormser et al. published an article in Clinical Infectious Diseases titled First Recognized Human Bite in the United States by the Asian Longhorned Tick, Haemaphysalislongicornis.

Picture taken by Jerry Zon while hiking. For more information: www.cdc.gov/Lyme

The heightened concern for the Asian long-horned tick is based on the fact that, in contrast to most tick species, a single female Asian long-horned tick can produce offspring (1,000-2,000 eggs at a time) without mating. As a result, hundreds to thousands of ticks can be found on a single animal, person, or in the environment. Livestock producers and pet owners should work with their veterinarians to maintain regular tick prevention and report any unknown tick species to their local department of agriculture.

Molecular Aspects of Measles Virus

  • Despite an Effective Vaccine, Measles Outbreaks Continue 
  • Genotyping by RT-qPCR Has Been Established by the CDC and WHO  
  • Improved Genotyping by Whole Genome Sequencing Looks Promising 

According to weekly updated data from the Centers for Disease Control and Prevention (CDC), the U.S. is currently experiencing the greatest number of measles cases reported since 1994. From January 1 to May 10, 2019, 839 individual cases of measles have been confirmed in 23 states. In some European countries and other parts of the world, the outbreak numbers are much greater. More than 100,000 people across Europe have been infected with the potentially deadly measles virus, which is spreading an ‘alarming’ rate, according to a recently reported statement by the World Health Organization (WHO). The WHO has said the rise in cases is ‘unprecedented’ for a preventable disease, and the United Nations children’s fund released a report revealing that every year for the last eight years, more than 20 million children have missed their measles vaccine.

Despite the ready availability of an effective vaccine, there are two major contributing factors to the occurrence of the current 2019 outbreak in the U.S. The first is misinformation about the safety of this vaccine, which is actually a “trivalent” formulation that provides protection against measles, mumps, and rubella (MMR vaccine) in a single injection. Because of misinformation, some parents are choosing to not have their children vaccinated, representing a group referred to as anti-vaxxers. In January, 2019, the WHO warned that “anti-vaxxers are now one of the greatest threats to world health.” Misinformation persists despite the widespread availability—online and otherwise—of reliable, factual information from the CDC and the WHO. 

The second factor is increased population mobility. In a society that travels for family reasons, business, pleasure, immigration, etc., the probability of contact with infected persons rises significantly. In any case, the increase in outbreaks in the U.S. is undeniable, and there are almost daily news reports on the quarantines that are imposed to prevent further spreading of this highly contagious disease. For example, this past April more than 900 students and staff members at two Los Angeles universities were quarantined or sent home, and in May, the Caribbean nation of St. Lucia quarantined a visiting U.S. cruise ship, barring any passengers or crew from leaving the boat while in port. 

As indicated by the title of this blog, I will focus on the molecular aspects of measles virus, rather than further commenting on efficacy, safety, anti-vaxxers, or how nations, states, or cities are attempting to cope with suppressing the spread of measles. Particular attention will be given to nucleic acids-based molecular methods that include virus identification by qPCR or sequencing. However, before getting to those topics, the next section provides some introductory information about measles.

About Measles

The CDC website for measles is a trove of reliable information, and is well worth checking out, as only some selected information is provided here. Historically, measles became a nationally notifiable disease in the United States in 1912, requiring U.S. healthcare providers and laboratories to report all diagnosed cases. In the first decade of reporting, an average of 6,000 measles-related deaths were reported each year. In the decade before 1963 (the year in which a vaccine became available), nearly all children got measles by the time they were 15 years of age. Each year then, among reported cases, an estimated 400 to 500 people died, 48,000 were hospitalized, and 1,000 suffered encephalitis (swelling of the brain) from measles. Symptoms of measles include a characteristic measles rash, as shown here.

In 1954, John F. Enders and Dr. Thomas C. Peebles isolated measles virus from the blood of 13-year-old David Edmonston. Nine years later, in 1963, John Enders and colleagues transformed this Edmonston-B strain of measles virus into a vaccine and licensed it in the U.S. In 1968, an improved measles vaccine developed by Maurice Hilleman and colleagues began to be distributed. This latter vaccine, called the Edmonston-Enders strain, has been the only measles vaccine used in the U.S. since then, and is administered as either trivalent MMR (see above) or tetravalent (MMR and varicella virus/chicken pox, MMRV) formulations. 

Measles is a highly contagious virus that lives in the nose and throat mucus of an infected person. It can spread to others through coughing and sneezing, but—thankfully—is not spread by any other animal species. Measles virus can live for up to two hours in an airspace where the infected person coughed or sneezed. If other people breathe the contaminated air or touch the infected surface, then touch their eyes, noses, or mouths, they can become infected. Measles is so contagious that if one person has it, up to 90% of the people close to that person who are not immune will also become infected. Hence the need for vaccinating a high percentage of the world’s population.

Genetic Analysis of Measles Viruses

Characterization: Measles is an enveloped negative-sense single-stranded RNA virus that needs RNA polymerase to form a positive-sense RNA, which then functions as viral mRNA for translation into proteins for the production of new virion materials. Wild-type measles viruses, as pictured here, are divided into eight clades (i.e. groups believed to have evolved from a common genetic ancestor) comprising a total of 24 genotypes based on the nucleotide sequences of their hemagglutinin (HA; aka H) and nucleocapsid (NC; aka nucleoprotein, N) genes, which are the most variable genes in the viral genome. The eight clades are designated A to H, with numerals (e.g. B2, B3, C1, etc.) to identify the individual genotypes. Within a genotype there may be multiple distinct genetic lineages. All vaccine strains are genotype A. 

This transmission electron micrograph (TEM) by the CDC/ Brian W.J. Mahy revealed the ultrastructural appearance of a virus particle, or ‘virion’, of the measles virus. Taken from publicdomainfiles.com and free to use.

The 450 nucleotides (nt) encoding the 150 carboxyl-terminal amino acids of the nucleoprotein have up to 12% nt variation between genotypes, and this entire 450-nt sequence is required for determination of the genotype. Measles virus genotyping can play an important role in tracking transmission pathways during outbreak investigations. Genotyping results can help confirm, disprove, or detect connections among cases. If two cases have matching 450-nt sequences, they are likely connected even if the connection is not obvious. 

Measles virus genotyping can help establish which foreign country may be the source of an imported U.S. case, since different genotypes circulate in different countries. However, genotyping alone is not sufficient, since each genotype can circulate in multiple countries and even in different regions of the world. Genotype data is therefore reviewed in conjunction with epidemiological information, such as travel and exposure histories, to determine which country may be the source of an imported US case.

Detection: The detection of measles RNA in a clinical sample can provide laboratory confirmation of infection. Real-time quantitative PCR (RT-qPCR) to detect measles RNA and endpoint RT-PCR to determine the genotype are performed at the CDC. These laboratory protocols are available upon request from the CDC. The RT-qPCR analysis, such as that pictured here, is more sensitive than the endpoint RT-PCR assay for detection of measles RNA in clinical samples, while the endpoint assay is routinely used to amplify the region of the measles genome required to determine the genotype.

RT-qPCR data for SYBR Green fluorescence analysis of five samples, each having three replicates. Taken from commons.wikimedia.org and free to use. 

To research this topic further, I used Google Scholar to find publications that covered “RT-qPCR (AND) measles” anywhere in the article. This gave more than 1,100 items, including a collaborative report by the CDC and the WHO titled Improving molecular tools for global surveillance of measles virus. The stated objectives of this study included the development of improved primers and controls for the RT-qPCR reactions used for genotyping of measles samples. The investigators found a new primer pair that was able to amplify the 450-nt sequence from measles viruses. These viruses represented 10 currently circulating genotypes, as well as a genotype A vaccine strain, and demonstrated 100-fold increased sensitivity compared to the previously used primer pair. A nested PCR assay further increased the sensitivity of detection from patient samples. 

It is worth interjecting here that improved PCR specificity and sensitivity of primer pairs can be obtained by using TriLink CleanAmp® PCR Products, which achieve “hot start” PCR conditions, thus avoiding the need to design and test different primer sequences or use nested primer pairs. Simply swap out the standard dNTPs or primers in your PCR assay for the corresponding CleanAmp® dNTP Mix or CleanAmp® Primers, respectively. Representative results, publications, and testimonials can be accessed at this link.   

Genome Sequencing of Measles Viruses

The 15,894-nt measles virus genome shown here schematically encodes six structural proteins: the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large polymerase (L) proteins. There are also two non-structural C and V proteins, which are both sub-products of the P gene: C results from an overlapping reading frame, and V from an edited transcript. Each coding region is preceded and followed by untranslated regions, of which the longest (1,012 nt) is the non-coding region between the genes for the M and F proteins (M/F NCR).

Schematic representation of the measles virus genome. The 15,894 nucleotides (nt) of the measles virus genome encode: nucleoprotein (N; 525 aa), phosphoprotein (P; 507 aa), matrix (M; 335 aa), fusion (F; 550 aa), hemagglutinin (H; 617 aa), large polymerase (L; 2,183 aa), C (299 aa) and V (186 aa) proteins. Coding regions of the genome (in white) are separated by non-coding regions (NCR; in grey). The longest NCR is that between the M and F genes: M/F NCR (1,012 nt). Amino acids = aa. Taken with permission from ©Penedos et al. PLoS One (2015) 10(11): e0143081.This is an appropriately credited open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium.

Penedos et al. state that, due to vaccination, the genomic diversity in circulating measles virus is decreasing. This means that the information provided by the 450-nt (aka N-450) genotyping “window” (see above) is increasingly proving insufficient in the description of outbreaks, as often little or no variation in the sequence is detected. To demonstrate interruption of measles circulation, countries must be able to distinguish between endemic transmission and importation events. The widening of the sequencing window for measles virus may then be necessary to verify measles elimination.

To investigate the possible utility of whole genome sequencing (WGS) as a means to provide this wider window, Penedos et al. studied over 60 samples, including 42 from the 2012 outbreak in the U.K., all of which were obtained from cell culture isolates or oral fluid specimens for NGS or Sanger sequencing. These samples, which represented measles virus genotypes D8, B3 and D4, were subjected to RT-PCR and the resultant amplicons were sequenced using NGS technology provided by Illumina (MiSeq instrumentation shown here). Sanger sequencing was used to close gaps in sequences. 

Illumina MiSeq instrument. Taken from Flickr and free to use.

Due to the technical complexity of the bioinformatic methods used for the NGS data processing, phylogenetic analysis, and epidemiological clustering, only the summary conclusions reported by Penedos et al. are given here:

  • Analysis of 32 whole genomes obtained from the outbreak indicates that the single nucleotide difference found between the two major groups of N-450 sequences detected during the outbreak is most likely a result of stochastic viral mutation during endemic transmission rather than of multiple importation events.
  • Phylogenetic analysis of each genomic region for the strains in this study suggests that most information is acquired from the non-coding region located between the matrix and fusion protein genes (M/F NCR) and the N-450 genotyping sequence.
  • Penedos et al. suggested that both M/F NCR and WGS could be used to complement the information from classical epidemiology and N-450 sequencing to address specific questions in the context of measles elimination.

Real-time Measles Species Identification by Nanopore Sequencing

Taken from Wikipedia and free to use.

Nanopore-based sequencing, which I have previously blogged about in various contexts, is now being used for real-time identification of various pathogens by virtue of its enablement of sequencing-by-scanning very long, single-molecules of DNA at very high speeds, as detailed elsewhere and depicted here in simplified cartoon form. Nanopore sequencing data starts to stream immediately, rather than being delivered in bulk at the end of a ‘run’. This enables an experiment to be stopped as soon as sufficient data has been gathered to answer the question being asked. It can also enable real-time selective sequencing, known as ‘Read until…’.   

I researched the literature to determine if real-time nanopore sequencing of measles virus has been reported, and was delighted to find that it has! Juul et al. have published a proof-of-concept investigation of a laboratory and analysis workflow in which, starting with an unprocessed (i.e. unenriched or unfractionated) sample, sequence data is generated and bacteria, fungi, and viruses present in the sample are classified to subspecies and strain-level in a quantitative manner. All of this is done without prior knowledge of the sample composition, and in only approximately 3.5 hours, which is quite remarkable to me. This workflow relies on the combination of the Oxford Nanopore Technologies’ MinION™ sequencing device and a real-time species identification bioinformatics software. 

It is important to emphasize that, unlike the methodology described in the previous section, this real-time pathogen classification does not use any pathogen-specific primers to selectively amplify one or more pathogens of interest for subsequent ensemble sequencing. Readers interested in the details can consult Juul et al. In a nutshell, they used authentic samples of a relatively large number of representative bacteria, fungi, and viruses—including measles virus, strain Edmonston—to prepare fragmented genomic DNA (for bacteria and fungi) and cDNA (for viruses). These fragments were converted into MinION™ sequencing libraries and sequenced for bioinformatics analysis. The analysis provided an easy to interpret summary report of the pathogens identified and their relative abundance, which was confirmed by RT-qPCR.

Concluding Comments

After reading published literature on nucleic acids-based molecular aspects of measles virus, from which I selected the above topics, I now have mixed feelings. On the positive side, there are safe and effective vaccines (MMR or MMRV) to provide protection against measles, and there are a variety of state-of-the-art PCR and sequencing methods to identify measles viruses for various scientific and health-related purposes. On the negative side, there is continued difficulty in getting high enough vaccination coverage in countries around the world to prevent breakouts, if not eradicate measles. How that problem may be solved is not obvious to me. Put another way, if you will, advances in the science of measles have been relatively straightforward, but widespread acceptance by global societies continues to be elusive.

As usual, your comments are welcomes.

Addendum

After writing this blog, a risk-based assessment study was published in The Lancet Infectious Diseases that provides a list of 25 counties in the U.S. where the next big measles outbreak is most likely to happen. 

Also, Larson and Schulz published and editorial in Science magazine titled Reverse global vaccine dissent. They briefly outline the history of vaccine dissent (i.e. anti-vaxxers), they state that, “[t]o mitigate the globalization of vaccine dissent, while respecting legitimate sharing of concerns and genuine questions, a mix of relevant expertise is needed. Technology experts, social scientists, vaccine and public health experts, and ethicists must convene and take a hard look at the different roles each group has in addressing this challenge. It needs everyone’s attention.”

May is Mental Health Month

  • Mental Health America Began This Annual Observance 70 Years Ago
  • Two Recent Studies Link Gut Microbe Composition to Mental Health Disorders: Clinical Depression and Schizophrenia  

Since 1949, Mental Health America (MHA) and its affiliates across the country have led the observance of May as Mental Health Month, reaching millions of people through the media, local events, and screenings. MHA welcomes other organizations to join it in spreading mental health awareness by using the May is Mental Health Month toolkit materials and conducting educational activities through #4Mind4Body. This blog is a way for TriLink BioTechnologies, on behalf of all Maravai LifeSciences businesses, to join MHA in “spreading the word” about this very important health issue.

In general, my blogs tend to revolve around topics trending in nucleic acids research. In keeping with this, I will briefly discuss two recent publications that provide new and compelling evidence linking human gut microbiome composition to depression and schizophrenia. These examples of the gut-brain connection (aka gut-brain axis) are especially noteworthy, since microbiome composition can be monitored by sequencing and altered by diet, as I have previously blogged about. Because of this, dietary modifications can be a healthy alternative to traditional medications, which have adverse side-effects and can be abused. 

Some Basics About Depression and Schizophrenia

According to the U.S. National Institute of Mental Health (NIMH), the lead federal agency for research on mental disorders, depression and schizophrenia are two among eleven major categories that include autism spectrum disorder, attention-deficit/hyperactivity disorder, bipolar disorder, etc. 

Depression: While there are a number of discrete forms of depression, all involve some type of serious mood disorder that affects how you feel, think, and handle daily activities, such as sleeping, eating, or working. To be diagnosed with depression, the symptoms must be present for at least two weeks. Depression (aka major depression or clinical depression) is one of the most common mental disorders, estimated by NIMH to afflict ~17 million adults in the U.S. 

Current research suggests that depression can be caused by genetic, biological, environmental, and/or psychological factors. It can happen at any age, but often occurs in adulthood, sometimes beginning as high levels of anxiety in children or adolescents. Even in the most severe cases, depression can be treated. The earlier treatment can begin, the more effective it is. Depression is usually treated with medications (antidepressants), psychotherapy, or a combination of the two. If these treatments do not reduce symptoms, electroconvulsive therapy and other brain stimulation therapies may be options worth exploring.

In regard to anti-depression medications and possible abuse, it is worth mentioning that, on March 5th of this year, the U.S. FDA announced approval of Spravato (esketamine) nasal spray, in conjunction with an oral antidepressant, for the treatment of depression in adults who have tried other antidepressant medicines but have not benefited from them (treatment-resistant depression). Spravato administration causes sedation and dissociation. Because of the risk of serious adverse outcomes resulting from these effects, and the potential for abuse and misuse of the drug, it is only available through a restricted distribution system.

Schizophrenia: This chronic and severe mental disorder affects how a person thinks, feels, and behaves. People with schizophrenia may seem like they have “lost touch with reality.” Schizophrenia is estimated to afflict ~2.5 million people in the U.S. Although not as common as other mental disorders, the symptoms can be very disabling, and usually start between ages 16 and 30. The symptoms of schizophrenia fall into three categories: positive, negative, and cognitive, which are further defined here. 

Scientists believe that although many different genes may increase the risk of schizophrenia, no single gene causes the disorder by itself. It is also believed that interactions between genes and aspects of the individual’s environment are necessary for schizophrenia to develop. Environmental factors may involve: viruses, malnutrition before/problems during birth, and psychosocial factors. An imbalance in the complex, along with interrelated chemical reactions of the brain involving the neurotransmitters dopamine and glutamate, may also play a role in the onset of schizophrenia.

Some experts believe that problems during brain development before birth may lead to faulty connections. The brain also undergoes major changes during puberty, and these changes can trigger psychotic symptoms in people who are vulnerable due to genetics or brain differences. Treatments typically involve antipsychotic medications and psychosocial therapy, both of which are discussed elsewhere. 

Links Between Gut Microbes and Depression

For more information on this complex topic, interested readers can consult a comprehensive review in 2014 by Mayer et al. titled Gut Microbes and the Brain: Paradigm Shift in Neuroscience, which covers preclinical literature for signaling between the brain and the gut microbiome. The possibility that alterations in the gut microbiome may play a pathophysiological role in various brain diseases, including depression, was considered by the authors to be speculative and in need of large population-based studies. The population size needs to provide statistically significant results upon which conclusions can be based.

Such a study has been recently highlighted in prestigious Nature magazine, in an editorial which notes that “just ten years ago, the idea that microorganisms in the human gut could influence the brain was often dismissed as wild. Not anymore.” As only a short synopsis of this landmark paper published in February 2019 in Nature Microbiology by Valles-Colomer et al. will be presented here, interested readers can consult the full publication at this link. Next-generation sequencing and bioinformatics methodologies used for identification and quantification of microbe diversity, which are technically quite complicated processes but now widely employed for many applications, can be read about by consulting the lead reference at this link.

In this study, Valles-Colomer et al. first assess gut microbiota compositional covariation with quality of life (QoL) indicators and general practitioner-reported depression in the Belgian Flemish Gut Flora Project (FGFP) population cohort (n = 1,054). The results were then validated both in the Dutch LifeLines DEEP (LLD) cohort with associated QoL and self-reported depression metadata (n = 1,063) and in previously published case–control studies on depression. To functionally analyze the gut microbiota neuroactive metabolic potential, they developed modules to profile the microbial pathways involved in neuro-microbiome mediator metabolism. Finally, application of these gut–brain modules in a shotgun-sequenced subset of the FGFP (n = 150) and validation in the LLD metagenomes data set (n = 1,063), and among a patient group suffering from treatment-resistant major depressive disorder (TR-MDD; n = 7), allowed Valles-Colomer et al. to link microbiota neuroactive capacity with QoL and depression.

The researchers found that two groups of bacteria, Coprococcus and Dialister, were reduced in people with depression. Gut–brain module analysis identified the microbial synthesis potential of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) as correlating positively with mental QoL, and indicated a potential role of microbial γ-aminobutyric acid (GABA) production in depression. However, it should be emphasized that these are correlations, and do not necessarily result in causation. 

 

DOPAC (PubChem Open Access) // GABA (PubChem Open Access)

According to the Nature editorial mentioned above, “the challenge now is to find out whether, and how, these microbe-derived molecules can interact with the human central nervous system, and whether that alters a person’s behavior or risk of disease. At least now, answering these questions is a wise pursuit, not a wild one.”

Links Between Gut Microbes and Schizophrenia

Almost concurrently with the above report in February 2019, Zheng et al. published a study in the prestigious Science Advances. This study also used next-generation sequencing and bioinformatics methodologies to link gut microbes with schizophrenia (SCZ). Sequencing reads from 63 patients with SCZ and 69 human controls (HCs) were clustered into 864 operational taxonomic units (OTUs) at 97% sequence similarity. A Venn diagram showed that 744 of 864 OTUs were detected in the two groups, while 56 and 64 OTUs were unique to patients with SCZ and HCs subjects, respectively.

To determine whether these altered OTUs were relatively specific to SCZ versus other neuropsychiatric disorders, they compared key differential bacterial taxa observed in SCZ and major depression. These findings indicated that the altered gut microbial composition observed in SCZ is specific relative to the gut microbiome changes observed in depression.

To identify key discriminative microbial markers, Zheng et al. performed a stepwise regression analysis based on the relative abundance of different gut microbes. This analysis showed that the most significant deviations between SCZ and HC subjects occurred for the bacterial families Aerococcaceae, Bifidobacteriaceae, Brucellaceae, Pasteurellaceae, and Rikenellaceae, which was said to suggest potential diagnostic value for SCZ.

In my opinion, the experimentally most interesting work involved fecal microbiota transplantation from patients with SCZ to germ-free mice to assess whether SCZ-relevant behavioral phenotypes might be linked with disturbed gut microbiota. Behavioral tests showed that mice transplanted with SCZ microbiota displayed locomotor hyperactivity, decreased anxiety- and depressive-like behaviors, and increased startle responses, suggesting that the disturbed microbial composition of SCZ microbiota recipient mice was associated with several characteristic of mouse models of SCZ.

Finally, to capture functional readout of microbial activity, Zheng et al. performed comparative metagenomic and metabolomic analyses of samples from the fecal transplanted-mice harboring “SCZ microbiota” versus “HC microbiota” to determine the potential mechanistic pathways by which the disturbed gut microbiota may modulate host physiology and behavior.

They identified decreased brain glutamate, disruptions in the glutamate-glutamine-GABA cycle, and altered amino acid and lipid metabolism as possible mechanistic targets that may underlie the altered behavior in the SCZ microbiota recipient mice, which was said to be consistent with known pathophysiology of SCZ. They also found significant changes in lipid species, especially involving glycerophospholipid metabolism, in the SCZ microbiota versus HC microbiota mice. This was said to be consistent with disturbances in serum and brain lipids previously observed in patients with SCZ, and with the fact that glycerophospholipids are major components of the myelin and neuronal membranes that are key regulators of synaptic function.

Concluding Comments

I hope that these brief synopses of the recent reports linking gut microbiota to clinical depression by Valles-Colomer et al., and to SCZ by Zheng et al., will in some way lead you to agree with me that such applications of nucleic acid sequencing to microbiome metagenomics are indeed representative of a new era for investigating mental health. My belief is that this type of sequencing will eventually lead to improved diagnosis of these and other neurological diseases. The extent to which controlled diets and/or transplants of therapeutic microbiota will contribute to improving mental health are still matters of speculation at this time. However, being an optimist, my outlook on these alternatives to traditional medications is favorable.

As usual, your comments are welcomed. 

Addendum

Although only indirectly related to the gut microbiome-brain focus of this blog, I think it’s worth mentioning the following findings recently published in Nature by researchers at the EMBL European Bioinformatics Institute and the Wellcome Sanger Institute.

1,952 uncultured candidate bacterial species were identified by reconstructing 92,143 metagenome-assembled genomes from 11,850 human gut microbiomes. These uncultured genomes substantially expand the known species repertoire of the collective human gut microbiota, with a 281% increase in phylogenetic diversity. 

Although the newly identified species are less prevalent in well-studied populations compared to reference isolate genomes, they improve classification of understudied African and South American samples by over 200%. These candidate species encode hundreds of novel biosynthetic gene clusters and possess a distinctive functional capacity that might explain their elusive nature. The researchers concluded that this work “uncovers the uncultured gut bacterial diversity, providing unprecedented resolution for taxonomic and functional characterization of the intestinal microbiota.”

Cell-Free DNA in Urine Developed for Early Detection of Liver Cancer

  • Exponential Increase in Publications on Cell-Free Biomarkers
  • Short Fragments of DNA in Urine Are Cancer Biomarkers
  • Rising Rate of Liver Cancer Prompts Earlier Detection by Urine Testing

Based on the large number of publications I found in PubMed, it is safe to say that investigations of “cell-free DNA biomarkers” have been on an upward trend in the field of nucleic acids. There has been an exponential increase in these publications during recent years, as shown in my chart below.

In my opinion, these articles revealed several broad technical categories such as diseases of interest, sources of samples (i.e. blood, feces, urine, tissue), methods of DNA isolation, and analytical methodologies. Importantly, these mostly academic or sometimes research-use-only applications are now transitioning towards becoming FDA-approved nucleic acid-based tests for in vitro diagnostics. An authoritative, up-to-date FDA list of these tests can be viewed here.

Many types of malignancies are included on this list, but despite its prevalence, liver cancer is not among them. Consequently, recent news about a promising candidate nucleic acid-based test for liver cancer using urine, which is arguably the most common and simplest of specimens to collect, prompted me to do some further research for this blog.

Urine and blood have become particularly interesting due to ease of access and safety compared to traditional, invasive biopsies. I have previously blogged about these non-invasive sample types in a post titled Sniffing Out Prostate Cancerin urine, and another post titled Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics.

Short Fragments of DNA in Urine Are Cancer Biomarkers

Although circulating DNA in the blood of cancer patients was detected and reported on four decades ago, such DNA was not investigated as a means of providing possible biomarkers until recent years, as reviewed by Anker et al. Urine is collected body fluid that has been filtered through the kidneys by nephrons, which are the microscopic structural and functional units of the kidney. Kidneys remove waste from the blood and return the cleaned blood back to the body. In 2000, Botezafu et al. reported that small amounts of cell-free circulating DNA in the blood can pass the kidney barrier into urine, and tumor-specific sequences can be detected in DNA isolated from urine.

In 2004, urine was first described as a useful source of circulating DNA for molecular diagnosis and prognosis. This landmark publication by a large multi-institutional team is titled Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. The corresponding author, Ying-Hsiu Su, is now with JBS Science (JBSS), a small startup company that followed up this initial observation with a publication in 2017 by Hann et al. titled Detection of urine DNA markers for monitoring recurrent hepatocellular carcinoma. A synopsis of this paper is featured later on in this blog, preceded by a short background on hepatocellular carcinoma (HCC).

HCC on the Rise and the Need for Early Detection

Credit: Kateryna Kon

According to a recent review by Ghouri et al., an epidemic of HCC, the most common malignancy of the liver, has spread beyond the predominance in Eastern Asian, and has been increasing in the Northern hemisphere, especially in the U.S. and Western Europe. It occurs more commonly in males in the fourth and fifth decades of life. Ghouri et al. add that, among all cancers, HCC is one of the fastest growing causes of death in the U.S. and poses a significant economic burden on healthcare. Chronic liver disease due to hepatitis B virus (HBV) or hepatitis C virus (HCV) and alcohol accounts for the majority of HCC cases. HCC is reportedly the fourth leading cause of cancer death worldwide.

According to Ghouri et al., the global burden of cancer in 2012 was an all-time high of 14 million cases, and that number is expected to grow to 22 million over the next two decades. Liver cancers have the seventh highest age-adjusted incidence rate in the world, with 0.8 million cases diagnosed for the year 2012.

Hann et al. note that HCC constitutes 70–85% of all types of liver cancer. The high mortality rate of HCC (wherein 85% of patients die within 5 years) is mainly due to late detection and a high recurrence rate. Rates of recurrence range from 15% for liver transplantation to nearly 100% for surgery or ablation. Recurrence is most common within 2 years.

Importantly, Hann et al. go on to provide the following statement in regards to the need for early detection of HCC:

MRI scan of the abdomen, liver cancer.

“Early detection of recurrent HCC has been difficult with the currently available diagnostic methods and serial imaging. Notably, there are no specific guidelines addressing how HCC recurrence should be monitored. Magnetic resonance imaging (MRI)/computed tomography (CT) imaging is the gold standard for diagnosis, although it is expensive and has limited utility in the detection of small tumors (< 2 cm), tumors in the presence of previously treated lesions (especially from local ablation), cirrhosis, obesity, and dysplastic nodules. Thus, there is an urgent unmet medical need to have a sensitive test for monitoring HCC recurrence.”

Early Detection of HCC Proof-of-Concept

Hann et al. then demonstrate the proof-of-concept (aka feasibility) of using urine for early detection of recurrent HCC by detecting three known HCC-associated DNA modifications: TP53 249T mutation (shortened TP53m), aberrant promoter methylation of glutathione S-transferase pi 1 (mGSTP1), and Ras association domain family 1 isoform A (mRASSF1A). These genes are then compared to the MRI imaging in a small (n = 10) blinded prospective study. These three DNA markers were chosen because of the reported availability of sensitive, cell-free DNA PCR assays for each of them:

  • TP53 in complex with DNA.

    Mutations in the TP53 gene are associated with approximately 50% of human cancers, as featured in a previous blog. A clamp-mediated PCR assay, followed by melting curve analysis, was available to detect TP53m. In this assay, the clamp suppressed 107copies of wild-type templates and permitted detection of TP53mtemplate, with a sensitivity of 0.1% (1:1000) of the mutant/wild-type ratio, assessed by a reconstituted standard.

  • For mGSTP1detection, an available methylation-specific PCR assay for the 5′-end region of the GSTP1 promoter targeted specific CpG sites with a forward primer, a reverse primer and a fluorogenic probe. Using this assay, mGSTP1 was detected with an assay sensitivity of 10 copies per reaction.
  • Quantitative methylation-specific PCR was similarly available for detection of

Interested readers can consult the aforementioned 2004 publication by Su et al. for extensive methodological details on how the low-molecular weight DNA in urine was isolated, as well as more information on the relationship of this DNA to circulating cell-free DNA and HCC tumor DNA.

Rather than try to paraphrase the conclusions of Hann et al., here is a quote of what they report:

“This study demonstrates the potential applicability of using urine DNA markers in combination with serum [alpha fetoprotein] AFP for the early detection of HCC recurrence in a small 10-case study. HCC recurrence is known to be the major factor for poor prognosis. In this small 10-case study, MRI identified recurrence in 5 out of 10 patients (cases 1–5). Encouragingly, for all 4 recurrent patients that remain in the study (cases 1–4), urine DNA markers were found to be elevated in urine samples as early as 9 months before MRI confirmation.

Although this is a small longitudinal 10 patient study, the potential of these urine DNA markers for management of HCC recurrence and important characteristics of HCC recurrence is demonstrated.”

“Bridge Award” by the National Cancer Institute for Commercialization

At the beginning of this blog, I made a passing reference to coming across recent news about a promising urine-based test for liver cancer. That news was actually a press release titled JBS Science, Inc. awarded $3 million bridge award by National Cancer Institute [NCI] to commercialize a urine test for liver cancer screening. While this press release led me to do the background research for the technical aspects described this blog, it also led me to discover that additional steps for commercialization will be carried out with funding by an NCI “Bridge Award,” which was new to me as a funding mechanism.

Credit: Lightspring. Modified by Jerry Zon

Since many of my readers are either in academia or small businesses, and seek support from the NIH, I thought it would be useful to look into the Bridge Award program. Briefly, the NCI’s Bridge Award is a specific type of Small Business Innovation Research (SBIR) funding to help small businesses bridge the funding gap between the end of an SBIR award and the subsequent round of financing needed to advance R&D toward a commercial product, as is visually depicted here.

Under the terms of the Bridge Award, SBIR-funded companies can apply for an additional $3M to cover both preclinical andclinical development costs. NCI Bridgeawardees are expected to obtain matchingfunds from investors, such as drug ordevice manufacturers, foundations, universities,or angel investors, for this 3-year award.

Since the Bridge Award must be matched by third-party investors, the NCI is providing a significant amount of “seed capital” and a not insignificant imprimatur, if you will, that together facilitate investments by third-parties.

The NCI provides a link to its SBIR Bridge Award “success stories,” which I found included numerous small companies covering various areas of cancer therapies, diagnostics, and cancer imaging technologies.

As usual, your comments are welcomed.

Addendum

After writing this blog, I decided to “deep dive” into databases for any information related to the novelty of a urine test for early detection of liver cancer, as it occurred to me that urine ought to be an obvious biofluid to look at for biomarkers.

I eventually found a lengthy review published in 2017 by Sengupta and Parikh titled Biomarker development for hepatocellular carcinoma early detection: current and future perspectives. I highly recommend this review to interested readers because it covers all types of biomarkers (i.e., proteins, DNA, RNA, and metabolites). Importantly, it discusses requisite phases of biomarker discovery that are necessary before a biomarker is appropriate for use in clinical practice.

As for the novelty of testing urine, a word search for urine in this comprehensive review by Sengupta and Parikh confirmed that the now NCI Bridge Award commercialized DNA-based urine test for early detection of HCC is indeed the first of its kind.

Recent Outbreaks of Acute Flaccid Myelitis (AFM) in Children Are Cause for Concern

  • AFM Is a Polio-Like Disease Confirmed in 24 States Across the U.S.
  • An Enterovirus Is Associated with AFM
  • AFM Has No Cure, but a Preventative Vaccine Appears Possible

About a month ago, a tweet about an AFM outbreak in children in Pittsburgh caught my attention. Because I had never heard of AFM, I searched scientific literature, learning that the disease is somewhat similar to polio, and has sporadically occurred in clusters around the world for quite a few years. One of the causative agents of AFM seems to be an enterovirus that mutates into various genotypes and phenotypes, based on information gathered from genome sequencing, PCR-based analyses, and clinical data. Now, combatting AFM and avoiding potentially major outbreaks are issues of growing concern.

This blog on AFM is my attempt—as a non-expert in virology—to distill complicated science and epidemiology into commentary that fits into what’s trending in nucleic acids research. Having said that, I’ll now start with a brief synopsis of AFM as a disease and its presumptive causative agent, “enterovirus D68” (EV-D68), and I’ll finish with some information on a possible protective vaccine against this debilitating illness.

Basic Facts About AFM

In general, my go-to sources for authoritative, up-to-date information on diseases are the National Institutes of Health (NIH) website, and the Centers for Disease Control (CDC) and Prevention website. Each web page is thoroughly referenced, with convenient links to primary sources. Here is a brief synopsis of information that I found on AFM by searching “Acute Flaccid Myelitis” on the NIH and CDC sites.

Child sleeping using an assisted-breathing device. iStock Credit: Juanmonino

Symptoms: AFM is a rare disease that affects the spinal cord, specifically the area of the spinal cord called gray matter. This causes the muscles and reflexes in the body to become weak. Symptoms of AFM include sudden (acute) weakness in the arm(s) or leg(s), along with loss of muscle tone (flaccid), and decreased or absent reflexes. In some cases, AFM can cause facial weakness, drooping of the eyelids, and difficulty swallowing, speaking, or moving the eyes. The most severe symptom of AFM is respiratory failure, which can happen when the muscles involved with breathing become weak. This can require urgent support and utilization of an assisted-breathing device, like the one pictured above.

Interested readers can access more information by perusing recent videos on AFM via YouTube. Most of these videos discuss a spike or outbreak of AFM in a particular state, and refer to AFM as a “polio-like” illness, an oversimplification that is widely used in mainstream media reports. Be forewarned that several posts among these AFM videos offer comments that I would politely describe as unscientific, if not plain bizarre.

Testing muscle response to nerve impulses using EMG. Credit: Romaset

Diagnosis: Most cases of AFM occur in children. Unfortunately for patients and parents, AFM can be difficult to diagnose because the symptoms are similar to other neurological diseases, such as Guillain-Barre syndrome (GBS), acute disseminated encephalomyelitis (ADEM), and transverse myelitis. Diagnosis may include an MRI of the spine, testing of the cerebral spinal fluid (CSF), tests checking nerve speed (nerve conduction velocity; NCV), and muscle response to nerve messages (electromyography; EMG). According to the CDC, as of October 31st 2018, there are 191 reported cases under investigation in 24 states across the U.S., of which 72 have been confirmed to be AFM cases.

Treatment: There is no specific treatment for AFM, but a neurologist specialized in treating brain and spinal cord illnesses may recommend certain interventions on a case-by-case basis. For example, neurologists may recommend physical therapy to help with arm or leg weakness caused by AFM. However, the extent of recovery varies – although some patients may make a full recovery, most have continued muscle weakness. The long-term outcomes of people with AFM remain unknown.

Causes: I was quite surprised at the CDC website’s statement that “AFM or similar neurologic conditions may have a variety of possible causes such as viruses, environmental toxins, and genetic disorders. Oftentimes, despite extensive lab tests, the cause of a patient’s AFM is not identified.” Given the sophistication of modern medicine and molecular diagnostics, I was expecting more definitive information. This in turn prompted me to research the reported virus-associated causes of AMF, which you can read about below.

EV-68 Is a Causative Agent for AMF

Enterovirus

Enteroviruses are a genus of positive-sense single-stranded RNA viruses, and they are named based on their transmission-route through the intestine (enteric means intestinal). A simplified depiction of the basic structural elements of an enterovirus is shown here. Classification of viruses can be inherently complicated, and it is not a topic that can be easily discussed due to the evolution of naming conventions, which sometimes appear to be a seemingly incomprehensible mix of Latin or Greek nomenclature, letters, and numbers.

Human enteroviruses are currently classified into 12 species: enteroviruses A through J (excluding letter I), and rhinoviruses A through C. Enteroviruses isolated relatively recently are named with a system of consecutive numbers. For example, EV-D68 belongs to enterovirus D. EV-D68 has a genome that contains a single open-reading frame, coding for a poly-protein (P1), the precursor of four viral capsid proteins, VP1, VP2, VP3, and VP4, and seven non-structural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. VP1 and VP3 are the major antigenic epitopes.

EV-D68 was first isolated in 1962 in California, from children with pneumonia and bronchiolitis. According to a June 2018 publication by Sun et al., there have been only 26 cases of documented EV-D68 respiratory disease in the U.`S. from 1970 to 2005. However, the upsurge of EV-D68 cases in the past few years showed clusters of infections in Europe, the Americas, Asia, Oceania, and Africa. In particular, more than 1,000 cases, including 14 deaths, were reported during an epidemic of EV-D68 infection in 2014 in the U. S., resulting in strong public attention toward this virus, as well as intensified research efforts on how to combat it.

A year later in 2015, Huang et al. reported carrying out a metagenomic shotgun sequencing protocol on clinical samples and negative controls, the results of which allowed for the assembly of 20 EV-D68 genomes: 6 complete, and 14 near-complete. A comparative genomic analysis revealed that EV-D68 strains circulating in the 2014 outbreak were significantly different from prior ones, and that they actually belong to a new clade. Importantly, two functional mutations in EV-D68 may alter its protease cleavage efficiency, thus leading to increased rate of viral replication and transmission.

In 2015, Zhuge et al. reported on the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (RT-PCR) developed by the CDC. Nasopharyngeal swab specimens from patients testing positive for rhinovirus or enterovirus were assessed using this EV-D68 RT-PCR, and the data were compared to results from partial sequencing analysis of the EV genome. The EV-D68 RT-PCR data showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. It was concluded that EV-D68 RT-PCR is a reliable assay for detection of EV-D68 in clinical samples, and it has the “potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.”

EV-D68 Is Spread in Multiple Ways

Although a number of whole-genome sequencing studies on EV-68 have now been reported in the context of comparative genomics, my inner “alarm bell” was triggered by a Lednicky et al. report. The researchers collected EV-D68 from classroom air using a filter-based air sampling method, and from environmental surfaces by swab sampling. Then, they amplified and sequenced the complete genome of the enterovirus. Here is a brief description of what they found and concluded:

EV-D68 was detected in 4-of-6 air sampler filters, and in 12-of-16 desk tops in a university classroom. cDNA synthesis was performed with avian myeloblastosis virus (AMV) reverse transcriptase and random hexamers on the viral nucleic acids extracted from filters or swabs, and PCR was performed using a panel of respiratory virus primers. Quantitative RT-PCR tests executed after the virus was identified indicated 400 to 5,000 genomic equivalents of EV-D68/m3 in the air samples. Viral RNA from the air sample with the highest concentration of virus was used for sequencing.

The researchers concluded that, “[a]s with our findings, high levels of airborne enteroviruses [have previously been] detected in a pediatric clinic, and this may be a common finding in indoor settings with enterovirus-infected individuals. Our work also suggests that young adults can produce airborne EV-D68 and raises the question of whether airborne transmission is important for spreading the virus.”

According to an article that I found in a journal on hygiene, EV-D68 can be found in bodily fluids such as saliva, mucous, sputum, and feces. It is transmitted through direct contact, including shaking hands, touching contaminated objects or surfaces, changing diapers of an infected person, or drinking water containing the virus. Following infection, the virus can be shed in stool for several weeks, and in the respiratory tract for up to 3 weeks. Shedding of EV-D68 can occur even in the absence of symptoms.

EV-D68 Vaccine Status

Vaccinating a baby.

The Benjamin Franklin axiom that “an ounce of prevention is worth a pound of cure” is as true today as it was when Franklin said it, especially when referring to modern vaccines. I researched the status of vaccines to protect against AMF, and found the following report by Dai et al.

These researchers describe the development of a virus-like particle (VLP)-based experimental EV-D68 vaccine. They found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1, and that they were visualized as spherical particles of ∼30 nm in diameter. Immunization of mice with EV-D68 VLPs resulted in production of serum antibodies that displayed potent serotype-specific neutralizing activities against EV-D68 virus in vitro.

Importantly, passive transfer of anti-VLP sera completely protected neonatal recipient mice from lethal EV-D68 infection. Moreover, maternal immunization with these VLPs provided full protection against lethal EV-D68 challenge in suckling mice. According to Dai et al., “these results demonstrate that the recombinant EV-D68 VLP is a promising vaccine candidate against EV-D68 infection.”

Hispid Cotton Rat (Sigmodon Hispidus).

Along similar lines, Patel et al. have found that cotton rats are permissive to EV-D68 infection without virus adaptation. Three different strains of EV-D68 studied all showed the ability to produce neutralizing antibody upon intranasal infection or intramuscular immunization. Patel et al. concluded that “our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.”

An EV-D68 mRNA Vaccine Seems Feasible

In 2017, a high-visibility Nature publication by Pardi et al. demonstrated that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP), encoding the pre-membrane and envelope glycoproteins of Zika Virus (ZIKV), elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination.

In my opinion, this landmark proof-of-concept study, which I’m pleased to say used modified mRNA comprised of 1-methylpseudouridine-5′-triphosphate obtained from TriLink, should be readily extended to EV-D68. The work of Dai et al. mentioned above, together with the information gained from genomic sequencing of EV-D68, provide the conceptual basis for synthesis of modified mRNA encoding one or more antigenic proteins as potential vaccines. These candidates could then be tested in one or both of the animal models mentioned above.

Unfortunately, drug and vaccine companies tend to be reluctant to invest in research targeting rare diseases. Development of a vaccine against AFM requires adequate financial resources. One can only hope that such resources become available, perhaps through a charitable foundation, the NIH, or maybe even crowdfunding, artistically depicted here.

The NIH has taken the lead on placebo-controlled clinical trials for a ZIKV vaccine; however, as detailed in a September 14, 2018 Science news article, a steep drop in Zika cases has undermined this and other planned related trials. This has led researchers to consider trials in which subjects are deliberately exposed to ZIKV, but studies like these would be faced with serious ethical and safety issues. Given the rarity of AFM cases, clinical evaluation of the efficacy of a candidate vaccine against AFM would encounter similar issues.

As usual, your comments are welcomed.

Footnote

After writing this blog, I became motivated to do a literature search for treatment of AFM. I found a 2017 publication by Tyler and coworkers titled Evaluating Treatment Efficacy in a Mouse Model of Enterovirus D68-Associated Paralytic Myelitis. This study evaluated 3 widely used empirical therapies for their ability to reduce the severity of paralysis in a mouse model of EV-D68 infection: (1) human intravenous immunoglobulin (hIVIG), (2) fluoxetine, and (3) dexamethasone.

Antibody binding to a virus.

Importantly, hIVIG, which was shown to contain neutralizing antibodies for EV-D68, reduced paralysis in infected mice and decreased spinal cord viral loads. Fluoxetine had no effect on motor impairment or viral loads. Dexamethasone treatment worsened motor impairment, increased mortality, and increased viral loads.

The following concluding statement from this 2017 publication is especially promising: “Results in this model of EV-D68-associated AFM provide a rational basis for selecting empirical therapy in humans.” In my opinion, of the 3 therapies investigated in this mouse model, treating patients with readily available hIVG seems to be the strongest course of action.

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Spotlight on TriLink Product Applications

  • Nearly 500 Publications in 2017 Cite Use of TriLink Products
  • Jerry Spotlights 20 Citing Oligos, Nucleotides, mRNA and Aptamers
  • 10 of These 20 Spotlighted Items Show Global Reach of TriLink Products

While thinking about possible topics to blog about, it occurred to me that researching recent publications on the applications of TriLink products would likely lead to many options. Using Google Scholar to do just that, I was given nearly 500 items, which is indeed plenty. However, choosing which to feature was neither an easy nor objective task. Having said that, and with sincere apologies to publications not spotlighted here, my “faves” and comments are given below, listed arbitrarily (not ranked) in four product categories: oligonucleotides, nucleotides, mRNA, and aptamers.

Taken from depositphotos.com

For convenience, each publication title can be clicked on to access the original article. Links to the cited TriLink products are also provided, alongside links to other adjunct information. Several trending “hot topics” and previous blogs are also noted.

Oligonucleotides

Taken from researchgate.net

Nucleotides

8-oxo-dGTP; taken from TriLink BioTechnologies // dPTP; taken from TriLink BioTechnologies

mRNA

Modified mRNA for new therapeutic approaches continues to be an amazingly hot area of R&D, which I have previous dubbed “modified mRNA mania” in a previous blog. Interested readers can peruse this link to ~300 items found in my Google Scholar search for TriLink and mRNA publications in 2017.

pseudo-UTP; taken from TriLink Biotechnologies // 2-thio-UTP; taken from TriLink Biotechnologies

Aptamers

2’-F-dCTP; taken from TriLink BioTechnologies // 2’-F-dUTP; taken from TriLink BioTechnologies

Global Reach

A pleasantly surprising aspect of the selected-product search results given above is the worldwide distribution of researchers using TriLink products. This global reach, if you will, is evident from the following countries outside of the USA, which I made point of mentioning:

The Netherlands, India, Austria, Switzerland, Turkey, Germany, Italy, Belgium, Republic of Korea, and Denmark.

All of the publications listed above were selected solely on the type of TriLink product used. Given the relatively small “sample size” of these selected publications, which are only 20-of-500, finding investigators in 10 countries outside of the USA is a compelling testimonial for the TriLink global reach.

World Science Day

Truth be told, when I was searching for a fitting image to visually convey the concept of “global science,” I came across the fact that the United Nations Educational, Scientific, and Cultural Organization (UNESCO) has designated November 10 as World Science Day, with an emphasis on peace and development. The stated intention is to highlight “the important role of science in society and the need to engage the wider public in debates on emerging scientific issues. It also underlines the importance and relevance of science in our daily lives.”

Taken from monitor.co.ug

According to UNESCO, “[t]he theme for 2018 is ‘Science, a Human Right’, in celebration of the 70th anniversary of the Universal Declaration of Human Rights (art. 27), and of the Recommendation on Science and Scientific Researchers. Recalling that everyone has a right to participate in and benefit from science, it will serve to spark a global discussion on ways to improve access to science and to the benefits of science for sustainable development.”

To me, this is a long-term objective which is indeed critical for betterment of future generations.

As usual, your comments are welcomed.

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FDA Approves First-of-a-Kind Test for Profiling Cancer Genes

  • Memorial Sloan Kettering Develops a Next-Generation Sequencing (NGS) Assay for 468 Genes
  • FoundationOne Does the Same for a 324 Gene Assay
  • Some View These Approvals as Tearing Down Conceptual Walls Between System Biology and Clinical Practices

Taken from jamanetwork.com (Credit: C. Lynm)

This blog’s title, which recently flashed around the globe as headline news, deserves to be echoed here, as it signals the achievement of a major milestone in nucleic acid-based diagnostics. Ever since the emergence of powerful PCR and sequencing methods for DNA analysis, there have been thousands of publications dealing with discovery and validation of genes associated with the genesis or signature of various types of cancer. A great deal of literature has also appeared regarding possible use of DNA analysis for cancer diagnostics.

Despite these continuing advances, the transition from research findings to doctors using DNA diagnostics has been hindered by the relatively long and costly process of gaining approval by regulatory authorities. In the U.S., the regulatory authority is the Food and Drug Administration (FDA). Readers interested in regulatory aspects can use this link to access FDA guidance for genetic testing in general. The focus herein is to provide a brief overview of what underlies the headline news echoed in the title of this blog.

Mutational Landscape of Metastatic Cancer

Molecular pathology of cancer has historically relied upon low-throughput approaches to interrogate a single allele in a single sample, such as those listed at this FDA website. By contrast, massively parallel “next generation” sequencing (NGS) has enabled a dramatic expansion in the content and throughput of diagnostic testing. However, the complexity of clinical NGS testing has prevented laboratories from achieving large-scale implementation, which is needed in order to maximize the benefits of tumor genomic profiling for large populations of patients. In addition, the clinical utility of mutation profiling requires evaluation of how molecular results are influencing therapeutic decisions in different clinical contexts.

To address this issue, the Memorial Sloan Kettering (MSK) Cancer Center developed a targeted tumor sequencing test, MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), to detect gene mutations and other critical genetic aberrations in both rare and common cancers. This test therefore detects all protein-coding mutations, copy number alterations, selected promoter mutations, and structural rearrangements in 341 (and more recently, 468) cancer-associated genes, as detailed by Zehir et al. This large team of investigators prospectively sequenced tumors from more than 10,000 cancer patients, who presented with a vast array of solid tumor types.

Taken Zehir et al. Nature Medicine (2017)

The DNA sample prep and analysis methodology described by Zehir et al should be appreciated as a tour de force of integrated automated sample handling systems. The method operates in conjunction with barcoded adapters, PCR, multiplexed DNA capture by biotinylated hybridization probes, and paired-end 100-base pair NGS reads – all to a mean depth of coverage of 718X.

Breakthrough FDA Approvals

On November 15th 2017, the FDA announced approval of MSK-IMPACT as a tumor profiling assay, i.e. an in vitro diagnostic (IVD) test, which can identify a higher number of genetic cancer mutations (biomarkers) than any test previously reviewed by the agency. The IMPACT test works by comparing a tumor tissue sample to a “normal” sample of tissue or cells from the same patient. This serves to detect genetic alterations that might help guide treatment options.

While the test is intended to provide information on cancer biomarkers, its results are not conclusive for choosing a corresponding treatment according to the FDA, which added that the assay is >99% accurate and capable of detecting a mutation at a frequency of ~5%. Detection of certain molecular changes (microsatellite instability) using the IMPACT test was concordant >92% of the time across multiple cancer types in 175 cases when compared to traditional methods of detection. Importantly, the Centers for Medicare & Medicaid Services (CMS) cover the cost of this test.

Shortly thereafter, on November 30th, the FDA announced approval of FoundationOne CDx (F1CDx) as an NGS-based IVD test that can detect genetic mutations in 324 genes and two genomic signatures in any solid tumor type. This test will also be covered by CMS. Additionally, based on individual test results, the new diagnostic can identify which patients with any of 5 tumor types may benefit from 15 different FDA-approved targeted treatment options. Its results provide patients and health care professionals all of this information in one test report, avoiding duplicative biopsies.

Tearing Down the Walls

A very recently published commentary by Allegretti et al. argues that, besides the many practice-changing implications, MSK-IMPACT and F1CDx approval by the FDA “tears down the conceptual walls dividing system biology from clinical practice, diagnosis from research, prevention from therapy, cancer genetics from cancer genomics, and computational biology from empirical therapy assignment.”

These authors further opine that that MSK-IMPACT and F1CDx have moral and ethical implications. For the first time, the FDA implies—and, the authors posit, some may say FDA plainly endorses the view—that “each patient at an advanced cancer stage has the right to have her/his cancer genome deciphered at the highest possible level of complexity compatible with current knowledge and technology, linking molecular information to state-of-the-art systemic therapies, as they become available.”

Therefore, according to Allegretti et al., extended NGS testing is becoming the standard of care in oncology. In the near future, “NGS profiling will likely be requested at progressively earlier stages, leading to a change in the engagement rules. No longer will the oncologist request a single assay for a single therapeutic option, e.g. BRAF or RAS mutational status for specific pathway blockade in specific cancers. On the contrary, it is implicit in [these] FDA approvals that the entire mutation catalogue will have to be made available to the medical team as soon as possible after diagnosis, and much before any specific therapy becomes applicable.” Pictorially, they view the new paradigm as follows:

Taken from Allegretti et al. J Exp Clin Cancer Res (2018)

I hope you will forgive my extensive quoting from Allegretti et al., but I believe it’s important to accurately convey the importance of their argument, given the profound impact of this type of thinking. Only time will tell the extent to which change become reality, as these innovations must deal with the hurdle of difficult dynamics between cancer care, cost containment, and societal conscience.

As always, your comments are welcome.

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The Scientist’s Top 10 Innovations in 2017—and Jerry’s Top Pick

  • Multiplexed Assays Are Trending
  • Miniaturization is Big—Pun Intended
  • Jerry’s Top Pick is an Amazing 4 Inch Cube

Taken from the-scientist.com

Welcome to my first blog of the New Year, 2018! My New Year’s resolution is to “double down” by giving my best effort to provide interesting and informative content about what’s trending in nucleic acid research. Having said that, and in keeping with tradition, this first blog of the year pairs my comments on the Top 10 Innovations in 2017, as reported by the The Scientist, with my personal “fav” for the best new product launched last year.

So, with an imaginary loud flourish of trumpets, read on to learn about The Scientist’s 10 winners that I’ve listed from 10th to 1st place—to build your interest—after which I comment on my personal fav.

Top 10 Innovations in 2017 Reported by The Scientist

10. TrueCut Cas9 Protein v2 from Thermo Fisher Scientific is a next-generation CRISPR-Cas9 protein engineered to deliver maximum editing efficiency across a range of genes and standard cell lines, as well as stem cells, T cells, and primary cells.

With regard to this product, I think it’s worth mentioning that TriLink offers mRNA-encoded Cas9, as an alternative to delivering Cas9 protein per se. Also, my several past blogs on CRISPR can be accessed here.

9. TSQ Altis Triple Stage Mass Spectrometer from Thermo Fisher Scientific robustly and reliably quantitates most analyte types, even in complex samples such as plasma and tissue, thus enabling wide applicability, including forensic toxicology and clinical research.

8. Chromium from 10x Genomics for profiling single-cell gene expression, enables deep profiling of complex cell populations, is provided as a complete droplet-based system of reagents, barcodes, hardware and software for sample prep prior to high-throughput sequencing.

7. Edit-R crRNA Library—Human Genome from Dharmacon provides users with an arrayed library of synthetic crRNA guides in a “one-well-per-gene” format, with four distinct guides per gene for redundancy to improve statistical power.

6. HiBiT Protein Tagging System from Promega is a new detection system for quantifying proteins in or on a cell of interest, using a small and easily integrated 11-amino-acid tag (HiBiT) that interacts with a complementary large 156-amino-acid component leading to bioluminescence.

5. SR-X Ultra-Sensitive Biomarker Detection System from Quanterix offers more than 80 different assays to test samples (e.g. blood, serum, cerebral spinal fluid, single-cell lysates) for cytokines or other markers of neurodegeneration or neuroinflammation, and more.

4. Blaze from Intabio is a system for detecting and identifying protein isoforms that aims to save pharma companies time in lab prep work for QC of biologics manufacturing. Launch will be “within the next few months” and “pricing is still yet to be set,” according to The Scientist.

3. QGel Assay Kit for Organoids from QGel provides fully synthetic extracellular matrix to reproducibly grow research organoids, which are miniaturized and simplified version of an organ produced in vitro.

2. i-STAT Alinity from Abbott is a handheld, cartridge-based blood-testing device for user-friendly point-of-care assays on a blood sample of just several drops—including glucose levels and hematocrit—with results directly delivered to a patient’s medical record for clinicians within 2-10minutes. Alinity is available in about 50 countries, but Abbott is waiting for a few more assays to be cleared by the US Food and Drug Administration before selling it stateside.

Regarding this product, I should mention that my several past blogs on point-of-care can be accessed here.

1. IsoCode Chip from IsoCode is new single-cell technology allowing researchers to characterize cells based on the proteins they secrete—as many as 42 different cytokines, chemokines, and other types of molecules. IsoCode chips contain thousands of long microchambers that house only single cells. Within each microchamber, 15 spatially separated slots contain up to three different antibodies targeting specific secreted proteins; upon binding, each antibody fluoresces in a different color to distinguish the proteins. This provides the ability to simultaneously profile thousands of individual T cells or immune cells at one time.

Taken from The Scientist Dec 2017

IsoCode chips come in 10 different panels, ranging from 24 to 42 antibodies per panel, at a cost of $500–$600. The automated IsoLight imaging and workflow platform can be purchased starting at $200,000. But the IsoCode chips can also be paired with other fluorescence microscopy systems.

Jerry’s Fav for the Best Innovation in 2017

My personal pick for this honor goes to the world’s first on-site Legionella DNA test to prevent Legionnaires’ disease, which was released this past November by the Canadian company Spartan Bioscience. According to a press release, it is the first on-site DNA test for Legionella bacteria and it can detect and quantify Legionella in only 45 minutes, compared to 10-14 days for off-site sample analysis using traditional culturing methodology. The system pictured here consists of a coffee-cup-sized portable DNA analyzer called the Spartan Cube, which employs a single-use disposable test cartridge.

Taken from ctvnews.ca

This innovative product stood out for me because it brings together the following topics that I have separately blogged about:

  • Reoccurring outbreaks of potentially fatal Legionnaire’s disease, such as that which recently hit New York City, and also shut down Disneyland.
  • Decentralized analytical testing for more rapid “sample-to-answer” applications on-site, i.e. in the field—wherever that might be—or at point-of-care in hospitals, clinics, and elsewhere.
  • Miniaturization and simplification of qPCR using cleverly engineered devices, such as the Spartan Cube, in conjunction with single-use disposable test cartridges.

Legionella is a common environmental bacterium that can infect the cooling towers of Heating, Ventilation, and Air Conditioning (HVAC) systems of large buildings. Infected cooling towers release aerosolized water droplets contaminated with Legionella into the surrounding air. Globally, there are hundreds of thousands of office-building towers, hospitals, hotels, shopping malls, and other large buildings at risk for infection by Legionella. Weekly testing with the Spartan system can rapidly detect Legionella bacterial growth early, and thus allow cleaning and decontamination of the cooling tower before Legionella reaches dangerous levels to human health.

In addition, and also importantly, traditional culture test methods can underestimate the Legionella concentration on site. The Centers for Disease Control and Prevention (CDC) found that Legionella culture can underestimate actual Legionella levels by a factor of 10 or more. Culture incorrectly reported that water samples were negative for Legionella an average of 11.5 percent of the time when in fact they were positive.

Paul Lem. Provided by Paul Lem

According to Paul Lem, M.D., CEO of Spartan Bioscience, who I contacted about cost to customers, “the price for the Cube and Legionella tests is $5-10K/building/year, depending on the building. It’s a subscription model.”

Regarding my further inquiry about testimonials to date, Dr. Lem provided a copy of a November 26, 2017 article reported in The Globe and Mail which quotes him as saying that “several property managers are testing the device at close to 100 properties, including BGIS and Ottawa’s KRP Properties, owned by tech entrepreneur [Sir] Terry Matthews.”

The article also states that “the market could be worth billions of dollars globally, encompassing office buildings, malls, hospitals, schools, theme parks, spas and so on. ‘Nobody really knows because the market doesn’t really exist yet,’” said Lem.

For the sake of increased public safety toward exposure to Legionella, let’s all hope that this application of the amazing Spartan Cube is indeed very successful. And, moreover, that 2018 is a great year for other nucleic acids-based innovations, many of which I look forward to blogging about here.

As usual, your comments are welcomed.

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Legionnaires’ Disease Outbreak in New York

  • First Identified as a New Pathogen 40 Years Ago, Legionella Persists
  • Legionella’s Life Cycle Involves “Biological Sanctuaries”
  • qPCR Proven to Outperform Antibody-Based Detection of Legionella

When I read about an outbreak of Legionnaires’ disease (LD) in New York City, baseball legend Yogi Berra’s famous quote, “It’s déjà vu all over again” immediately came to mind, along with the irony of Berra playing for the New York Yankees. So, if you’re much younger than me, you’ll likely not know why “It’s déjà vu all over again” and you may wonder who Berra was. You can read about him later elsewhere, but for now you should read on to learn about Legionnaires disease and why déjà vu is apropos.

History of LD

Notable positive events during 1976 in the United States included our Bicentennial Celebration, unveiling by NASA of the first space shuttle (the Enterprise), establishment of Apple Computer Company by Steve Jobs and Steve Wozniak, and Silly Love Songs by Paul McCartney and Wings ascending to #1 on the charts. While many of these events were the beginning of fabulous things to come, one proved to be the beginning of something catastrophic. American Legionnaires who gathered in Philadelphia, Pennsylvania for the Bicentennial were struck with a mysterious epidemic of fatal respiratory disease.

Taken from networks.org

Sadly, 182 members of the Pennsylvania American Legion were affected, and 29 individuals died after they returned from the convention in Philadelphia. The epidemiological and microbiological studies continued for months before scientists began to understand what had happened. Much of the basic framework of our knowledge of Legionnaires disease, as the epidemic came to be known, was developed by a team from the CDC and the Pennsylvania Department of Health, as detailed elsewhere.

Taken from case1study.wikispaces.com

The cause of the disease remained a mystery until 1977 when an investigative team led by J. E. McDade and C. C. Shepard (of the Leprosy and Rickettsia Branch, Virology Division, Bureau of Laboratories, CDC) reported on the isolation of a Gram-negative bacillus found in patient samples. As often done for naming pathogens after sources, the genus of this rod-shaped bacterium was aptly named Legionella. Legionella includes the species L. pneumophila, which caused the pneumonia-like illness medically named legionellosis, but commonly referred to as LD.

2017 LD Outbreak Hits New York City—Again

In June of this year, forty years after the first characterization of Legionella, it’s lethal infectivity reoccurred in an outbreak in the Upper East Side of the Manhattan borough of New York City, leaving one person dead and six other people sickened. According to a newspaper account, this outbreak occurred within 11 days, and may have been triggered by contacting contaminated water as has happened in other cases.

While this incident affected relatively few people compared to other previous outbreaks, including one in the Bronx borough of New York City in 2015 that killed 15 people and sickened more than 70, it’s a scary reminder of the persistence of Legionella in the environment. In this regard, it has been reported that 200 to 400 cases of the illness are recorded each year in New York, despite the monitoring of 6,000 water systems wherein Legionella can flourish in warm conditions. This environmental factor provides a segue into what genomic sequencing has revealed about Legionella.

Genomics-Based Insights on Legionella

The bacterial pathogen L. pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause severe pneumonia associated with Legionnaires disease. As detailed elsewhere, recent advances in genome sequencing has had a major impact on understanding of the pathogenesis, evolution and genomic diversity of Legionella.

A lipopolysaccharide cell wall and several outer membrane proteins are essential virulence factors. Central to the pathogenesis of L. pneumophila is its Type IV secretion system, which translocates over 270 effector proteins into the host cell, thus allowing this bacterium to manipulate host cell functions to its advantage and assures intracellular survival and replication.

Within aquatic media, as depicted below, Legionella exist as part of biofilms, which provide a protective environment—or biological sanctuary, if you will—wherein the bacteria exhibit marked increase in resistance to biocidal compounds and chlorination. Aside from the resultant difficulty of purging water systems to be free of Legionella, these bacteria can invade and multiply within protozoa (which are ubiquitous and include amoeba), thus providing yet another biological sanctuary. Protozoa are present in all aquatic or moist environments, and can be found in even the most inhospitable parts of the biosphere, thus providing further protection to Legionella.

Taken from Comas Nature Genetics (2016)

The actual infectious particle is not known but may include excreted legionellae-filled vesicles, intact legionellae-filled amoebae or free legionellae that have lysed their host cell. Transmission to humans occurs via mechanical means, such as air-conditioning units, taps and showerheads, as well as others listed by the World Health Organization (WHO).

Infection in humans occurs by inhalation of the infectious particle and establishment of infection in the lungs. After ingestion by macrophages, L. pneumophila have been found to inhibit acidification and maturation of its phagosome. Following a 6–10 hour lag period, the bacteria replicate for 10–14 hours until macrophage lysis releases dozens of L. pneumophila progeny.

It’s worth noting that, according to WHO, there is no direct human-to-human transmission of Legionella, which in my opinion is why incidence of LD remains relatively low.

Gardening Can Be Bad for Your Health—No Joke.

Unfortunately, there are other ways of contacting LD besides ingestion of tainted water. At the risk of sounding flippant, gardening can be seriously bad for your health because of contracting LD by breathing in aerosolized Legionella from contaminated soil. This is especially true in New Zealand, which has the highest incidence of LD in the world, according to a recent publication, with L. longbeachae being the most clinically relevant species. This infectious agent is predominantly found in soil and composted plant material. Most cases occur over spring and summer, and the people at greatest risk are those involved in gardening activities.

Taken from lawrieco.com.au

Some agricultural experts advocate smelling soil to assess its quality, stating that “[t]he smell of a soil can often reveal its state of health, sweet or offensive or plain bland,” and adding “the smell does not actually come from the dirt itself, but from soil microbes that inhabit a healthy soil environment. Sweet smelling soil has good levels of organic carbon which is vital to supporting the world of billions of beneficial bacteria and fungi in every cup of healthy soil.”

These soil sniffing experts, however, fail to consider the presence of pathogenic organisms including L. longbeachae. I, for one, will carefully avoid purposefully smelling any soil when gardening, and will instead be sure to wear a good mask capable of filtering out aerosolized Legionella, as you should too!

Nucleic Acid-Based Detection of Legionella

Rapid and effective diagnosis of LD is extremely important so that timely and appropriate therapy can be provided, thereby lowering the morbidity and mortality rates and reducing the health and economic costs associated with this disease. Surprisingly, diagnosis is reportedly established solely by time-consuming microbiological tests. Luckily, it looks like testing procedures could soon change for the better, thanks to PCR and NGS.

Taken from corisbio .com

Earlier this year, Christovam et al. assessed the accuracy of various detection tests in patients suspected of being infected with Legionella and in patients with laboratory-confirmed LD. Investigators analyzed urinary Legionella antigen detection, direct fluorescent antibody (DFA) staining, serological testing and PCR vs. culture analysis (the reference standard). The sensitivity and specificity for PCR were 83 % and 90 %, respectively, whereas DFA sensitivity and specificity were 67 % and 100 %, respectively. Moreover, PCR had high sensitivity and specificity for early diagnosis of LD.

Taken from letsfixit.co.uk

While the study results reported by Christovan seem promising, less definitive results have been reported. Krøjgaard et al., who compared culture and qPCR assays for the detection of Legionella in 84 samples from shower hoses and taps in a residential area before and after two decontaminations. Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc. was said to be “dubious.” However, the rapid detection of high concentrations of Legionella by qPCR was said to be valuable as an indicator of risk, although it may be false positive compared to culture results. Detection of a low number of bacteria by qPCR was said to be a strong indication for the absence of risk.

Not surprisingly, the advent of powerful next-generation sequencing (NGS) is emerging as a better method for genus-specific, sensitive and quantitative determination of Legionella. In 2017, Pereira et al. reported findings from a study using NGS to differentiate 20 pathogenic strains of Legionella in fresh water systems. A genome standard and a mock community consisting of six different Legionella species demonstrated that the reported NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. Comparison of quantification by real-time PCR showed consistency with the NGS data, thus indicating that NGS “provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method.”

Concluding Comments

Aside from providing a brief introduction and update on LD, my additional intent was to alert readers—without undue alarm—to the myriad circumstances in which Legionella can infect humans. According to the aforementioned list provided by WHO, the most common form of transmission of Legionella is inhalation of contaminated aerosols produced in conjunction with water sprays, jets or mists. Infection can also occur by aspiration of contaminated water or ice, particularly in susceptible hospital patients.

Researching transmission of Legionella in Google Scholar led me to find additional information (see links below) that you may find useful or interesting.

Thankfully, as I’ve said before, Legionella is not transmitted human-to-human. The scary aspect of Legionella, however, is that it’s continually mutating, which raises the specter of emergence of a strain that can spread within a human population. Let’s hope that this doesn’t happen and/or that modified mRNA vaccines can be quickly produced to combat that possibility.

As usual, your comments are welcomed.

Addendum

After finishing this blog, there was a Reuters news report on October 9, 2017 that Michigan’s top medical official, Dr. Eden Wells, will be charged with involuntary manslaughter for her role in the city of Flint’s water crisis, which was linked to an outbreak of LD that caused at least 12 deaths. Dr. Eden Wells would become the sixth current or former official to face involuntary manslaughter charges related to this crisis, which principally involved lead contamination in the city’s water supply.

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