Patent Docs

Patent Luminaries Try to Set Congress Straight on Drug Price Controls

By Kevin E. Noonan --

Over the past few years the drumbeat regarding the cost of healthcare in general and drugs in particular has steadily mounted (see "Faux-Populist Patent Fantasies from The New York Times").  Patents are often (and quite wrongly) blamed for the purported high costs, despite the crucial role patents play in spurring innovation; indeed, some advocates have been accused of skewing their statistics to make their arguments appear plausible (see "Unreliable Data Have Infected the Policy Debates Over Drug Patents"; "Tillis Wants More Info on I-MAK and Other Data Driving Anti-Patent Narratives Around Drug Pricing"; and "I-MAK Defends Integrity of Its Patent Data in Response to Tillis Letter").  But a recent tactic is for many, including members of Congress, to argue that the Bayh-Dole Act, and in particular the so-called “march-in rights” provisions (Bayh-Dole Act, Pub. L. No. 96–517 (1980), Section 203) entitle the Federal government to set drug prices, at least for those drugs developed using Federal grant money for their discovery or development (and despite valiant efforts by Joe Allen and the Bayh-Dole Coalition to explain how their reading of the Act is in error; see www.bayhdolecoalition.org).  Alternatively, the provisions outlining the jurisdiction of the Court of Federal Claims in cases involving patents and copyrights (28 U.S.C. § 1498) have been similarly invoked as giving the Federal government the right to set drug prices because the drugs in question are patent-protected.

Today, a letter was sent by 25 scholars, former judges, and former government officials to the Senate Health, Education, Labor and Pensions Committee, the Chair and Ranking Member of the House Ways and Means Committee, and to Secretary of Health and Human Services Xavier Becerra "correcting false claims that the federal government can use the Bayh-Dole Act or § 1498 to impose price controls on prescription drugs."  That letter is set forth below and deserves serious consideration by the Biden Administration, Congress, and the rest of us to prevent unwise attempts to misapply the Bayh-Dole Act, as set forth eloquently and specifically in the letter.

A Better, CRISPR World Assayed in The New York Times

    By Kevin E. Noonan --

Idealism is a wonderful and at the same time frustrating character trait, because the world is not ideal as it is and is unlikely to ever be, but the motivation to achieve a more ideal world (or at least a more equitable one) is universal in human cultures (if only as a hope for a better world for our descendants).  A professor at the University of California/Berkeley, Fyodor Urnov (at right), recently expounded on the dream of using CRISPR gene editing to cure disease in a New York Times article and addressed the real-world challenges and obstacles faced in trying to use this breakthrough technology broadly enough for that ideal world to come a bit closer to reality (and for a change the NYT did not invoke patent protection as the all-purpose bogeyman thwarting such more idealistic outcome).

Dr. Urnov begins his essay with real-life examples of the types of genetic diseases faced by many (albeit a minority) of Americans (that statistical demographic playing a significant role in the story he is telling), in children and young adults, either from birth or in sudden onset or as a ticking timebomb of consequences determined by their faulty genes.  The initial promise of "DNA fixes" was gene therapy beginning in the 1980's, as the fruits of the revolution in molecular biology and the identity of genes responsible for these diseased began to be elucidated.  While there have been some successes in these efforts, the mechanisms for achieving them (usually involving virus-mediated gene insertion into an affected cell or tissue) have been both uncertain and "jaw-droppingly expensive" as Dr. Urnov relates (citing the $3.5 million price tag for Hemgenix, CSL Behring's cure for Hemophilia B).

An improvement with tremendous promise is CRISPR (which stands for "clustered regularly interspaced short palindromic repeats" in homage to how it was initially found in bacteria).  As Dr. Urnov explains, CRISPR technology can specify repair of a genetic mutation "right where the 'typo' occurs" in an affected gene.  He recites the recent uses of CRISPR technology for providing treatment for congenital blindness, sickle-cell disease, certain heart diseases, nerve disease, cancer, and HIV.  These successes engender in Dr. Urnov the hopeful prospect that CRISPR-based genetic medicine could be achieved in future for a variety of ailments, wherein its genetic specificity could provide directed and tailored cures for many diseases.  His vision for a future child afflicted with genetic disease is that:

A dedicated CRISPR ‌cures ‌‌center at a university-affiliated hospital . . . takes the diagnosis [of a genetic disease] and morphs it into an order form for a manufacturing facility to create the medication that will repair the faulty gene.  After a month of testing and data review by hospital clinicians and university scientists, the physician does a simple IV injection of the resulting CRISPR medicine, and after a three-day stay at the hospital to confirm‌ the gene editing went according to plan, the child is sent home.

He cites several examples, in the U.S. and abroad, of CRISPR successes and companies like CRISPR Therapeutics, Vertex, Intellia Therapeutics, and Regeneron who have achieved them in recent years, with other examples of on-going work by other companies.

But then Dr. Urnov explains the realities that create real impediments to this genetic utopia.  There are 7,000 known genetic diseases (caused by a defect in a single gene) and 400 million people worldwide affected by them.  While he posits a simple experimental path from diagnosis to treatment, he also acknowledges that this would be "only the first step in a four-year journey likely to cost at least $8 million to $10 million."  The first reason for this situation is the regulatory requirements in the U.S. and Europe aimed at "ensur[ing] safety and efficacy of the experimental medicine," Dr. Urnov laying out the time- and money-consuming path from beginning preclinical studies to actually producing the "CRISPR medicine," which itself is subject to well-deserved demanding specifications.  These efforts can cost more than $1 million and take years when animal testing is included in the calculus.  As a result, the hypothetical child having a genetic disease amenable to CRISPR treatment "stands little chance of timely treatment," he writes.  Added to the complexities of bringing the CRISPR drug to market, Dr. Urnov recognizes that the investment can easily be over $10 million and could (in some instances) be capable of treating only a single patient (for idiosyncratic mutations not shared by a class of patients such as with sickle cell disease where a particular shared mutation converts a glutamic acid residue to valine in the hemoglobin protein).  And the doctor notes that many patients do not have the luxury of time for all these processes to play out, making the theoretical possibility of the CRISPR magic bullet even more frustrating and tragic.

There also have been examples of actual therapies (in "conventional" gene therapy) that have hit the roadblock of investment failure by private companies trying to commercialize university-created inventions (one involving UCLA being mentioned specifically in Dr. Urnov's article).  But while there is an economic justification for the cost of these drugs (cited here, that "a one-time cure saves the health care system years of costly supportive care"), "[f]or diseases with fewer than 100 patients, such prices [$2-3 million per patient] are still not enough for these efforts to make commercial sense."  The doctor cites cases where companies have discontinued clinical studies due to the cost of bringing the drug to market, and the dim or at least insufficient prospects of a return on investment, that have made the economic proposition untenable.

Dr. Urnov proposes that as a first step towards improving this situation is for the regulatory regime to be revised; while it makes sense for diseases having tens of thousands of patients for a proposed treatment, for "one-of-a-kind genetic typo" cases there should be a "streamlined" process (akin to what was employed for CAR-T therapies in their infancy).  As for the role of who develops and pays for the treatments, he recognizes that private biotechnology companies cannot bear the burden.  "Tapping into federal and state funding could provide a path forward," Dr. Urnov posits, citing recent clinical trial collaborations between UCLA, UCSF, and UC/Berkeley for a gene-editing approach to curing sickle cell disease.

But recognizing the zeitgeist he asks:  "Why should the average taxpayer contribute to building medicines for rare diseases?  Would the money be better spent on finding treatments for common ailments?"  His answer is that helping people with rare diseases will foster development of CRISPR-based treatments for more common ones.  He acknowledges that, for now, ethical considerations will limit CRISPR therapies to those patients with diseases like cancer and "devastating genetic ailments."  He expresses hope that development of these treatments will eventually produce in genetic medicine the types of advances that have been achieved in other industries.  And he advocates that while "[u]nless things change dramatically, the millions of people CRISPR could save will never benefit from it," "[w]e must, and we can, build a world with CRISPR for all."

This essay raises a fundamental ethical question, should we and must we do what we can to achieve CRISPR-based and other revolutionary treatments in the face of the economic realities that there is no short-term economic justification for them?  History is replete with examples of seemingly fruitless efforts (Columbus, the space program) that turned out to have unappreciated (or at least unexpected) benefits, economic and otherwise.  The long-term view is that developing CRISPR and other medical technologies will produce a more robust, more productive populace, which will inure to our benefit and well-being.  The question Dr. Urnov raises is whether we will have the vision to leave behind the short-term view of present-day dollars and cents and see and act upon that possibility for a better, healthier world.

President Biden's Biotechnology and Biomanufacturing Executive Order: Funding

    By Kevin E. Noonan --

President Biden announced his signing of an Executive Order launching a major effort to enhance U.S. capabilities in biotechnology and biomanufacturing last week (see "President Biden Signs Executive Order on Biotechnology and Biomanufacturing Innovation").  The White House also held a Summit on Biotechnology and Biomanufacturing last week to discuss how these efforts will be funded (accompanied by a Fact Sheet).

The summit, led by National Security Advisor Jake Sullivan, Director of the National Economic Council Brian Deese, and Director of the Office of Science and Technology Alondra Nelson, Ph.D., was attended by various biotechnology and biomanufacturing stakeholders who spoke to the importance of the initiative and the funding it will provide for its stated goals.  Also participating were Secretary of Health and Human Services Xavier Becerra, Secretary of Energy Jennifer Granholm, Deputy Secretary of Defense Kathleen Hicks, Deputy Secretary of Agriculture Jewel Bronaugh, Under Secretary of Commerce for Standards and Technology and Director of the National Institute for Standards and Technology Laurie Locascio, and Director of the National Science Foundation Sethuraman Panchanathan, as well as Senator Mark Warner (D-VA) and Representative Deborah Ross (D-NC, 2nd).

This funding, amounting to a $2 billion commitment of Federal money, included:

• $40 million from the Department of Health and Human Services to expand the role of biomanufacturing for active pharmaceutical ingredients (APIs), antibiotics, and the key starting materials needed to produce essential medications and respond to pandemics.

• $1 billion from the Department of Defense over five years to improve the bioindustrial domestic manufacturing infrastructure over five years to catalyze the establishment of the domestic bioindustrial manufacturing base that is accessible to U.S. innovators.

• Also from the Department of Defense is $270 million over five years to commercialize research and "support the advanced development of bio-based materials for defense supply chains, such as fuels, fire-resistant composites, polymers and resins, and protective materials."

• And also from the Department of Defense is $200 million to "support enhancements to biosecurity and cybersecurity posture" for biomanufacturing facilities and defense supply chains.

• $500 million from the Department of Agriculture to support "independent, innovative, and sustainable American fertilizer production to supply American farmers" using biotechnology and biomanufacturing advances.

• $100 million from the Department of Energy for R&D for conversion of biomass to fuels and chemicals, including R&D for improved production and recycling of biobased plastics.

• Another $60 million from the Department of Energy to "de-risk" scale-up of biotechnology and biomanufacturing to increase commercialization of biorefineries that produce renewable chemicals and fuels that significantly reduce greenhouse gas emissions from transportation, industry, and agriculture.

• $20 million from the National Nuclear Security Administration in the Department of Energy to "advance U.S. capabilities to anticipate, assess, detect, and mitigate biotechnology and biomanufacturing risks, and . . . integrate biosecurity into biotechnology development."

• $14 million from the Department of Commerce for the National Institute of Standards and Technology (NIST) for biotechnology research programs to develop measurement technologies, standards, and data for the U.S. bioeconomy.

In addition to these specific funding announcements, the National Institutes of Health is expanding its biotechnology entrepreneurship bootcamp (the I-Corps program); the National Science Foundation will implement its previously announced competition for funding of regional Innovation Engines; and the FDA will attempt to use regulatory science, technical guidance, and increased engagement with industry to support advanced manufacturing.

Of course, these commitments reflect current budgets and (to some extent) discretionary spending for these agencies and Executive branch Departments.  Whether and the extent to which President Biden's vision encompassed by the Executive Order will come to fruition will depend on further funding from Congress in the regular budgetary process, and those decisions cannot be other than politically motivated.  In view of the propensity for support or opposition to government policies such as this one to depend on whether the administration is on the "right" side of the aisle illustrates the truth in the adage that elections have consequences, even for programs aiming to enhance and improve the country's competitiveness in important areas like biotechnology and biomanufacturing.

Penguin Genetics Suggests Complex History

By Kevin E. Noonan --

Penguins are unique among bird species, having lost the ability to fly more than 60 million years ago and adopting a "hyperspecialized marine body plan" consistent with their unique habitat in the higher latitudes of the Southern Hemisphere.  Key geological events are believed to be in part responsible, with major climate oscillations that led the penguins to find ecological refuge and then recolonize previous niches.

A recent study by an international team of scientists* reported specifics of penguin natural history and genetic structure in a paper entitled "Genomic insights into the secondary aquatic transition of penguins," in the journal Nature Communications (13: 3912 (2022).  A significant difference between this study and earlier studies is that this one includes fossil penguins, which make up almost 75% of all penguin species (earlier studies were limited to mitochondrial genes or a few nuclear genes) "combining genomes from all extant and recently-extinct penguin lineages (27 taxa) (Table 1), stratigraphic data from fossil penguins (47 taxa), and morphological and biogeographic data from all species (extant and extinct)."

The data from these analyses support the origin of penguins in New Zealand, originating in stem penguins with extensive local radiation followed by dispersing to Antarctica and South America in multiple invasions of these environments.  Crown penguins, on the other hand, originated in South Africa -- 14Mya from these migrations, followed by dispersal back to New Zealand at least three times, with two of these inferred migrations occurring before the prevailing Antarctica Circumpolar Current had been established (west to east, which would have facilitated these migrations).  The origin of these species coincides with global cooling in the middle Miocene; during at least some of the time periods relevant to penguin evolution there were no polar ice sheets.

These researchers found "incongruities" between phylogenetic trees created by genetic versus conventional morphology-based phylogenies.  They found evidence that rapid speciation of Crown penguins was associated with incomplete lineage sorting (ILS) (due in part to the persistence of polymorphisms across different speciation events) or introgression (transfer of genetic material between related species) resulted from 5% ILS content within ancestors of several penguin species (Spheniscus, Eudyptula, Eudyptes, and several subgroups within Eudyptes).  These results are consistent with closely related penguin species can hybridize in the wild and "provides a temporal framework for this rapid radiation: the four extant Spheniscus taxa are all inferred to have split from one another within the last ~3 Ma, and likewise the nine extant Eudyptes taxa likely split from one another in that same time."

This observed pattern of relatively recent divergence of penguin species within the past 3 million years is a consistent feature among penguin lineages and is correlated with post-speciation introgression events between these populations (events not detected between species that are geographically disparate).  These researchers found "a genomic signature of a period of physical isolation during the Last Glacial Period (LGP) with increased climate fluctuation and environmental uncertainty, followed by postglacial contact and introgression as Earth warmed once again"; similar evidence can be seen in other marine taxa during this period, where penguins and other species moved north away from the South Pole and then back as the earth warmed.

The authors also analogize their historical evidence of climate change during this period with the effects of man-made climate change recently, stating that taxa capable of migration survived better than taxa that were "residential" and foraged close to shore.

Using the integrated evolutionary speed hypothesis (IESH) (which assesses the effects of temperature, water availability, population size, and spatial heterogeneity) these researchers concluded that penguins have the slowest evolutionary rate of all bird species, something these scientists attributed to their "increasing aquatic ecology."  Paradoxically perhaps this perceived evolutionary slowdown could be seen in crown penguins over the first 10 million years of its history but then an uptick in evolutionary rates around 2 million years ago, correlating with the glacial-interglacial cycles during that time has something to do with it.

Turning to genes associated with penguin evolution, these scientists report correlations between phenotypic adaptations (including "their locomotory strategy, thermoregulation, sensory perception, and diet").  They identified three classes of genetic sources of these adaptations:  positively selected genes (PSGs); rapidly evolving genes (REGs); and pseudogenes.  These results showed 15 PSGs and six REGs were penguin-specific adaptations (Fig. 4a) below, five genes containing penguin-specific substitutions, seven pseudogenes, and two gene expansions, illustrated in this figure:

Three of these genes (TBXT and FOXP1, relating to cartilage, tendons, and limb bone and SMAD3, encoding transforming growth factor 3 are related in penguins (and other flightless birds) to "shortening, rigidity, and increased density of the forelimb bones."  Also implicated in limb formation and adaptations was TNMD, a PSG, which the authors speculate may be the cause of "wholesale replacement of penguin distal wing musculature by tendons."  And the authors note that KCNU1 and KCNMA1 genes related to calcium sequestration have been expanded in the genomes of penguins (as well as grebes).

Other genes are implicated in phenotypic features such as densely-packed waterproof feathers, thick skin, and a layer of subcutaneous fat which enables the animals to thermoregulate in cold environments.  These genes include APPL1, TRPC1, EVPL, wherein the APPL1 and TRPC1 genes are involved in glucose levels and fatty acid breakdown through adiponectin; the authors hypothesize these genes promote white adipose fat storage which provide both insulation and an energy reserve.  Another important adaptation is the capacity to withstand hypoxic conditions, and in this regard the transferrin receptor 1 gene TFRC was found to be under positive selection pressure (consistent with expression of this gene in an oxygen-dependent manner even in domesticated cattle).  The FIBB and ANO6 genes, which are involved in blood coagulation, were found to be selected for in species having the capacity for the deepest dives (>500m).  Hemoglobin genes HBA-αA (A140S) and HBB-βA (L87M) had the noted penguin-specific amino acid sequence variations found conserved in all penguin species as were modification in myoglobin genes.  Finally, in this regard a gene that may help widen blood vessels to decrease blood pressure during deep dives, TRPC4, was found to be a PSG.

Vision, particularly low-light vision, is another phenotype hshowing penguin-specific adaptations.  It was known that penguins have only three functional cone photoreceptor types, and the green cone opsin gene RH2, was found in all penguin species to have a 12bp deletion encoding a lysine residue critical for chromophore binding.  These researchers also found specific genetic alterations in red cones associated with blue-shifting the pigment and facilitating a shift in optimum wavelength consistent with ambient underwater light (and having implications for foraging under such conditions).  And deactivation of CYP2J19, a gene that encodes a carotenoid ketolase responsible for producing red oil droplets in avian cone, the authors surmise "likely allows for higher retinal sensitivity when foraging in dim light conditions, as seen in nocturnal owls and kiwis."  Finally, other genes thought responsible for visual sensitivity at low light levels (including TMEM30A (PSG), KCNV2 (REG), CNGB1, and GNB3) show evidence of selection and/or mutations specific to penguins.

Diet is another aspect of penguin lifestyle associated with genetic changes.  This implicates penguin taste sensitivities, and these authors report that penguins have retained the capacity to taste only sour and salty flavors (and the genes associated with these capacities); the genes for sweet, bitter and umami have been lost in all penguin species.  Curiously, according to the authors, the penguin genome contains no active chitinase genes and only one pseudogene, which is inconsistent with a diet comprising crustaceans (because many species are known to consume large amounts of these animals.  The researchers propose (based on fossil evidence) that penguins lost their chitinase genes during ~50 million year interval where they did not consume this prey.

Immune-related genes were also assessed, and 51 PSGs were detected for such genes.  The authors speculate that the selective pressure may be the result of host-pathogen co-evolution.  Examples of such genes include bacterial-recognizing Toll-like receptors TLR4 and TLR5 and within these genes site proximal to the lipopolysaccharide-binding site of TLR4 and the flagellin-binding site of TLR5.  A gene involved in viral RNA detection, IFIT5 is also under positive selection in penguins with positively selected amino acid sites in the protein analogous to similar sites in the human ortholog of this gene.  A similar pattern of selection was found in the penguin gene encoding glycoprotein-mediated hepatitis C viral entry (CD81) with a cluster of positively selected amino acid sites that correspond to similar sites in humans.  Finally, the transferrin gene has undergone positive selection in penguins, which the authors speculate reflects resistance to Corynebacterium infection and diphtheritic stomatitis which are known to cause increased chick mortality.

The authors conclude that penguins acquired most of the genetic adaptations associated with their unique (for birds) aquatic habitat early in their speciation from other birds and that the strong positive selection for these traits is responsible for the low amount of evolutionary change in all penguin taxa.  But their strong adaptation to the colder climate in the peri-South Pole region puts them at risk in the current warming climate trends.  Accordingly these authors suggest that the canary in the global warming coal mine might actually be a penguin.

*Researchers from Demark, Norway, France, the UK, Germany, various European countries, the United States, Argentina, South Africa, New Zealand, China, and Australia reported the results of their studies in a paper entitled "Genetic Insights into the Secondary Aquatic Transition of Penguins," Nature Communications 13: 1-13

The Further Elucidation of Domestic Dog Ancestry

By Kevin E. Noonan --

An international cadre of scientists* from almost 70 institutions worldwide recently reported their findings in the scientific journal Nature that the domesticated dog (Canis familiaris) arose from two populations of ancestral grey wolves (Canis lupus) (see "Grey wolf genomic history reveals a dual ancestry of dogs").

The grey wolf is recognized as the species that survived the last Ice Age (the last glacial maximum or LGM occurring ~28,000-23,000 years ago) to give rise to domesticated dogs, but little is known about progenitor wolf populations.  Siberian grey wolves are known to have survived from this period but it is unknown the extent to which other grey wolf populations, which were widely distributed in the Northern Hemisphere for the last few hundred thousand years, either went extinct or responded to changes in climate by adaptation.  Archeological evidence places domesticated dog populations arising ~14,000 years ago, and divergence from grey wolf species occurring ~40,000-14,000 years ago.  But analyses of modern grey wolves and dogs have been unable to resolve domesticated dog origins due to, inter alia, dog genetic diversity and local extinction and gene flow occurring after domestication.

To attempt to address these deficiencies, the researchers assessed 72 ancient wolf genomes over the past 100,000 years from Europe, Siberia, and North America:

Sites of ancient wolf genomes reported in the study

(consisting of 66 newly sequenced genomes, 5 previously sequenced specimens, and one ancient dhole genome from the Caucasus, dated to be more than 70,000 years old and used as an "outgroup" control).  These samples were ~69% males and through mitochondrial DNA analysis averaged 21,573 ± 5,133 years old.  As explained in the paper, the researchers "merged single-nucleotide polymorphism (SNP) genotypes called from these genomes with those from worldwide modern wolves (n = 68), modern (n = 369) and ancient (n = 33) dogs, and other canid species [and t]he total dataset spans the last 100,000 years."

The results of genetic analyses on these SNPs showed a directionality of gene flow in wolf populations, from the Siberian wolf populations to the European and Central Asian wolves and not vice versa, starting after a time less than ~23,000 years ago; these conclusions were supported by mitochondrial DNA assessments ("our results suggest that Siberia acted as a source and Europe as a sink for migration throughout the Late Pleistocene and show no evidence of gene flow in the other direction").  Paradoxically, these results also showed that there remains what the researchers termed a "minority fraction of deep European ancestry" that has persisted in modern wolf populations, having 10-40% of their ancestry that is more divergent than the oldest Siberian wolves studied.  Some of this diversity was identified as coming from the African grey wolf in the Near East and an unknown canid originating in Tibet.  This persistent evidence indicated to these researchers that grey wolves, unlike other megafauna (e.g., woolly mammoths) did not come close to extinction.  And some North American grey wolf populations show evidence of admixture with coyotes, species that began to diverge from one another ~700,000 years ago (no similar coyote-wolf admixture was seen in Eurasian grey wolf populations).  The genetic relationships between these populations were represented in the paper by this graph:

And while there is some evidence of Siberian grey wolf genetic admixture in North American wolf populations the genetic evidence reported by these authors showed no reverse admixture of North American grey wolf populations into Siberian grey wolves.  The evidence also suggests local extinction in North American grey wolf populations during the LGM of the last Ice Age.

In comparing the proportion of genetic variation between rather than within grey wolf populations, the researchers found that wolf populations showed low levels of variation even between populations in distant regions in the Pleistocene.  In the last ~10,000 years, however, (i.e., the Holocene) further gene flow from Siberia was not detected while gene flow from European populations was seen in modern wolves from China and Siberia, with population bottlenecks perhaps (the authors speculate) being due to human "persecution" in the past few centuries (supported by widespread effective population size declines).

In further analyzing the observed connectivity in grey wolf populations throughout the past 100,000 years the authors report assessment of alleles at specific genetic loci, finding twenty-four regions in grey wolf genomes having some evidence for natural selection.  The observed connectivity between grey wolf populations resulted in mutations becoming fixed in these population between 40,000 and 30,000 years ago in the case of the IFT88 gene (a gene wherein disruption results in craniofacial abnormalities and cleft lips in mice and humans) on wolf chromosome 25, a characteristic shared with domesticated dogs.  (It might be recalled that changes in visage in domesticated dogs has been hypothesized as being relevant to their acceptance by humans; see "Selection for Facial Features in Domestic Dogs: The Evolution of Cuteness").  Additionally, these researchers reported that "[t]hree regions with evidence for selection overlap olfactory receptor genes, with variants on chromosome 15 increasing in frequency from close to 0% to 100%" between 45,000 and 25,000 years ago, "suggesting that olfaction was a recurrent target of adaptation in wolves" and that "[m]ost of the detected selection episodes occurred before the divergence of dogs, and dogs share the selected alleles."  Illustrating how natural selection and intentional breeding can operate on similar phenotypic determinants, the authors report that "a region on chromosome 10, where variation among dogs is associated with body size, drop ears and other traits [has been] under recent selection in specific dog breeds" was also found to have been selected in wolves in the last 20,000 years.

While the impetus for the reported research was to better understand the origin of domesticated dog species from earlier wolf populations, the results of these studies showed that history is more complex than anticipated.  A closer relationship was observed between modern domesticated dogs from eastern Eurasian wolves than western Eurasian populations, but modern domesticated dogs in the Near East and Africa could trace their ancestral species from a population of grey wolves related to extant southwestern Eurasian populations.  Recent population genetic events, including admixture and population changes further complicate the picture, according to the paper.  As summarized, the authors state that "[t]hese results could be taken to support an eastern or central Eurasian dog origin outside of north-eastern Siberia, but we cannot draw firm geographical conclusions in the absence of ancient wolf genomes from these and other candidate regions."

To further complicate matters, their results showed that "dogs have variable proportions of two distinct components of wolf ancestry" between Siberian wolf progenitors and European grey wolf populations, as illustrated by this diagram:

The authors state that the data were not sufficiently robust to distinguish between there having been an independent domestication event from these population or from admixture of domesticated dogs with local wolf populations.  As a consequence, the researchers failed to find a direct match between either of these ancestral species and these two sources of modern domesticated dogs, concluding that "the exact progenitor populations remain to be located."

*Anders Bergström, David W. G. Stanton, Ulrike H. Taron, Laurent Frantz, Mikkel-Holger S. Sinding, Erik Ersmark, Saskia Pfrengle, Molly Cassatt-Johnstone, Ophélie Lebrasseur, Linus Girdland-Flink, Daniel M. Fernandes, Morgane Ollivier, Leo Speidel, Shyam Gopalakrishnan, Michael V. Westbury, Jazmin Ramos-Madrigal, Tatiana R. Feuerborn, Ella Reiter, Joscha Gretzinger, Susanne C. Münzel, Pooja Swali, Nicholas J. Conard, Christian Carøe, James Haile, Anna Linderholm, Semyon Androsov, Ian Barnes, Chris Baumann, Norbert Benecke29, Hervé Bocherens, Selina Brace, Ruth F. Carden, Dorothée G. Drucker, Sergey Fedorov, Mihály Gasparik, Mietje Germonpré, Semyon Grigoriev, Pam Groves, Stefan T. Hertwig, Varvara V. Ivanova, Luc Janssens, Richard P. Jennings, Aleksei K. Kasparov, Irina V. Kirillova, Islam Kurmaniyazov, Yaroslav V. Kuzmin, Pavel A. Kosintsev, Martina Lázničková-Galetová, Charlotte Leduc, Pavel Nikolskiy, Marc Nussbaumer, Cóilín O'Drisceoil, Ludovic Orlando, Alan Outram, Elena Y. Pavlova, Angela R. Perri, Małgorzata Pilot, Vladimir V. Pitulko, Valerii V. Plotnikov, Albert V. Protopopov, André Rehazek, Mikhail Sablin, Andaine Seguin-Orlando, Jan Storå, Christian Verjux, Victor F. Zaibert, Grant Zazula, Philippe Crombé62, Anders J. Hansen, Eske Willerslev, Jennifer A. Leonard64, Anders Götherström, Ron Pinhasi, Verena J. Schuenemann, Michael Hofreiter, M. Thomas P. Gilbert, Beth Shapiro, Greger Larson, Johannes Krause, Love Dalén & Pontus Skoglund.

Ticked Tabby Cats and Their Genetic Bases Elucidated

By Kevin E. Noonan --

The domestic cat has been the subject of much study, recently involving its genetic structure, genomic DNA sequence, and comparisons with other felines.  The first such study was published in 2014, when an international effort led by Stephen J. O'Brien at the Oceanographic Center, Nova Southeastern University, Ft. Lauderdale, Florida reported the complete genomic sequencing of the domestic cat, Felix catus.  The report, entitled "Annotated features of domestic cat – Felis catus genome," was published in GigaScience 2014, 3:13 (August 5, 2014) (see "Domestic Cat Genome Sequenced").  The study reported sequencing of a female Abyssinian cat named Cinnamon, a mixed-breed cat from Russian named Boris, and Sylvester, a wildcat ancestor of domestic cats.  The report showed that domestic cats have retained "a highly conserved ancestral mammal genome organization" in comparison with ancestral cats (see Driscoll et al., 2007, "The near eastern origin of cat domestication," Science 317: 519–23).  Both species, F. catus and Felix silvestris, have 38 chromosomes, 18 pairs of autosomes, and two pairs of dimorphic gender-determining chromosomes.  Details of the domestic cat genome structure included the presence of 217 loci of endogenous retrovirus-like elements (amounting to 55.7% of the entire genome, comprised of long interspersed elements (LINEs), short interspersed elements (SINEs), satellite DNA, retroviral long terminal repeats (LTRs) and "others"); 21,865 protein coding genes (open reading frames or ORFs), detected by comparison with eight mammalian genomes (from human, chimpanzee, macaque, dog, cow, horse, rat, and mouse); and a wealth of genetic variability in single nucleotide polymorphisms (SNPs), insertion/deletion events (indels); novel families of complex tandem repeat elements; and short terminal repeat (STR) loci.

Since that time more individual cat genomic sequences have been determined and assembled in the 99 Lives Cat Genome Consortium.  These efforts resulted in a more comprehensive elucidation of the feline genome and insights into genetic bases for disease.  A paper published in the Public Library of Science, entitled "A new domestic cat genome assembly based on long sequence reads empowers feline genomic medicine and identifies a novel gene for dwarfism," PLoS Genetics 16(10): e1008926 on October 20, 2020, reported a revisit of the genomic sequence of Cinnamon, an Abyssinian breed domestic cat previously sequenced.  Rather than focusing on one cat, this group* performed whole genome sequencing (WGS) of 54 domestic cats and aligned the sequences to detect single nucleotide variants (SNVs) and structural variants (SVs).  As a consequence of these studies, sequences comprising the ~300,000 gaps in the annotated sequence reported to the Cinnamon Abyssinian were obtained, to produce a new reference cat genome denoted in relevant databases as Felis_catus_9.0.  This genome comprised 2.84 gigabasepairs (Gb), of which only 1.8% (1.38 megabasepairs, Mb) was not assigned to a specific chromosomal location.

While such assessments of global genetic structure are informative, analysis of genes resulting in well-known phenotypes have been slower to arrive.  One such study was published in March 2021 in Animal Genetics 52: 321-32, entitled "Mining the 99 Lives Cat Genome Sequencing Consortium database implicates genes and variants for the Ticked locus in domestic cats (Felis catus)."  In this paper, scientists from the U.S. and Australia* reported the genetic basis of the Ticked tabby coat pattern, a phenotype whose genetics long have been used empirically by cat breeders.  (Amathematically, the Consortium database contains genetic information from 195 individual cats.)

Three autosomal alleles at a single genetic locus in wild and domestic cats are understood to control the tabby coat pattern: Abyssinian (T^a, also known as "ticked"); mackerel (T^m, aka striped); and blotched (t^b, aka classic, blotched) (where these allelic abbreviations follow the convention that capital letters indicate Mendelian dominant traits and lower-case letters are inherited as recessive genes); the blotched phenotype was designated as the "classic" tabby by Linnaeus in 1758.  Various combinations of these alleles result in complicated coat patterns involving the legs, head/face, tail, and torso.

Failure of this understanding to explain cats that have spotted coats, like Egyptian mau and ocicat, induced further genetic analyses that uncovered at least three other loci involved in coat pattern; these include Tabby and Ticked, where the Tabby locus affects the mackerel and blotched patterns.  Located on the cat A chromosome, the candidate gene encoded by this locus, laeverin (LVRN) or transmembrane aminopeptidase Q, Taqpep, encodes 'a membrane-bound metalloprotease and plays a regulatory role in extravillous trophoblast migration."  The genetic bases for coat patterns involving the Tabby locus were found to be even more complex, involving genes in a pigment switching signaling pathway, and can have a phenotype having individual hairs in the coat pattern that alternates between melanin types that are seen illusorily to be brown.

The other locus, the Ticked locus, had been localized on the cat B1 chromosome but the gene residing at the locus had not been determined until this report.  The famous Cinnamon had the ticked pattern and accordingly her DNA was used by these researchers in one strategy as having the "reference" allele and in another as having the "variant" allele in performing their search for the gene or genes at the Ticked locus.  Of the 195 cats in the 99 Cat Consortium database, 80 had either solid or white coats and their ticked-tabby phenotype could not be determined (regardless of their underlying genotype).  Among the cats whose genomic DNA was assessed were two obligate heterozygous ticked cats identical by descent for their ticked allele as they are parent–offspring bred from a ticked Somali"; three known Abyssinians, including Cinnamon; and three other cats showing the ticked phenotype.

The researchers used eight short tandem repeats that segregated with the Ticked phenotype as identified by genome scanning methods to find a 17 centoMorgan (cM) linked region on cat chromosome B1 for their analysis of candidate variants.  Seven phenotype filters were applied (e.g., "eliminate intergenic, intronic and synonymous variants; (ii) consider the variant zygosity for the [whole genome sequencing entry for Cinnamon;" etc.) to identify increasingly rare variants in this region of the B1 chromosome; after filtering only one variant remained, located in the gene for Dickkopf Wnt Signaling Pathway Inhibitor 4 (DKK4).  The protein encoded by this gene is known to be "a member of the dickkopf (Dkk) family of cysteine-rich secretory proteins that are antagonists of Wnt signaling pathways, involved in antero-posterior axial patterning, limb development, somitogenesis and eye formation."  These researchers report finding a G>A transition mutation at position 188 of this gene, resulting in a Cys63Tyr amino acid sequence change in the encoded protein.  The researchers also reported finding at lower frequency another transition mutation (C>T) in this gene, resulting in a change at amino acid 18 from Ala to Val.  The position of these mutations is shown in the following Figure:

These researchers further report that protein structure modeling suggests that these mutations disrupt "a key disulfide bond" in a "cysteine-rich domain" in the first mutation or a signal peptide cleavage site in the Dkk protein in the second mutation.  The resulting change in protein conformation for the first mutation is illustrated in the following Figure:

In either case the researchers conclude that disrupting the function of this protein (as the consequences of these mutations suggest) results in the observed ticked phenotype.

The authors recognize that the ticked phenotype is rare in outbred cats (and the observed allele frequency, wherein no other cats from the 195 cats in the database have this allele, is consistent with its rarity) and dominant, suppressing patterning and resulting in cats having no discernable coat pattern.  But they also recognize the search for genes that influence coat pattern in cats is not concluded, stating:

Additional genotyping of the proposed variants, in a large cohort of phenotyped cats, as well as supportive functional data, would clarify the role of these variants in cat coat pattern development.  The identified variants do not clarify the pathways leading to the production of the spotted coat phenotype in cats, suggesting that additional genes influence other tabby patterns in domestic cats.  The allelic series for the Ticked locus is suggested as Ti^A = Ti^CK > Ti⁺, where the Ti^A allele represents the p.Cys63Tyr variant and the Ti^CK allele represents the p.Ala18Val variant.

* From the Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri – Columbia, Columbia, MO and the School of Health and Behavioural Sciences, University of the Sunshine Coast, Sippy Downs, Australia; the paper in an appendix credits the hundreds of researcher who have contributed to the 99 Live Cat Genome Consortium.

Further and More Detailed Study of Domestic Cat Genome

By Kevin E. Noonan --

The advent of technology making feasible elucidation of whole genomic sequencing over the past 30 years has led to reports of many if not most important or interesting animal genomes (including the most celebrated results of the Human Genome Project) (see, e.g., "Nautilus pompilius Genome Determined"; "Giraffe Genome Reveals Relevant Adaptations"; "Avocado Genome Elucidated; Durum Wheat Genome Revealed"; "Rose Genome Reveals Its Exquisite Complexities"; "Silver Birch Genetics Explained"; "Genomic Sequence of Strawberry Determined"; "Koala Genome Sequenced"; "Tomato Genome Determined"; "Lowland Gorilla Genome Sequenced"), as well as important historical relationships newly appreciated (see, e.g., "Did Neanderthal DNA Persist in Modern Humans as a Defense against Xenobiotic Viruses?"; "Chicken Origins Established (But Philosophical Questions Remain)"; "Genetic Analyses of Sweet Potato Genome Sheds Light on Speciation and Global Dispersion Patterns"; "The Domestication History of Apples Revealed by Genomic Analysis"; "Evidence of Geographic Change in Central America from Genome Studies of Eciton Ant Species"; "Dolphin Genes Show Relationships between Large Brains and Energy Metabolism Similar to Humans and Elephants").

But what has become evident recently is that most of these genomes were in one way or another incomplete, due typically because there were regions of this or that genome that were resistant to accurate sequencing or come other biologic idiosyncrasy.  The most recent example of this reminder of the complexity of biological organisms is our old friend the domestic cat, which built (as most of the new studies do) on the earlier genomic elucidations.  In this case, the earlier study in question was published in 2014, when an international effort led by Stephen J. O'Brien at the Oceanographic Center, Nova Southeastern University, Ft. Lauderdale, Florida reported the complete genomic sequencing of the domestic cat, Felix catus.  The report, entitled "Annotated features of domestic cat – Felis catus genome," was published in GigaScience 2014, 3:13 (August 5, 2014) (see "Domestic Cat Genome Sequenced").  The study reported sequencing of a female Abyssinian cat named Cinnamon, a mixed-breed cat from Russian named Boris, and Sylvester, a wildcat ancestor of domestic cats.  The report showed that domestic cats have retained "a highly conserved ancestral mammal genome organization" in comparison with ancestral cats (see Driscoll et al., 2007, "The near eastern origin of cat domestication," Science 317: 519–23).  Both species, F. catus and Felix silvestris, have 38 chromosomes, 18 pairs of autosomes, and two pairs of dimorphic gender-determining chromosomes.  Details of the domestic cat genome structure included the presence of 217 loci of endogenous retrovirus-like elements (amounting to 55.7% of the entire genome, comprised of long interspersed elements (LINEs), short interspersed elements (SINEs), satellite DNA, retroviral long terminal repeats (LTRs) and "others"); 21,865 protein coding genes (open reading frames or ORFs), detected by comparison with eight mammalian genomes (from human, chimpanzee, macaque, dog, cow, horse, rat, and mouse); and a wealth of genetic variability in single nucleotide polymorphisms (SNPs), insertion/deletion events (indels); novel families of complex tandem repeat elements; and short terminal repeat (STR) loci.

The report also contained an extensive comparison between domestic cats and other species ("reference genomes") in terms of gene numbers, using genes with the longest mRNA and corresponding coding sequences (click on table to expand).

More recently, further results of applications of improved genomic sequencing methods and technologies have provided a more comprehensive elucidation of the feline genome and insights into genetic bases for disease.  A paper published in the Public Library of Science, entitled "A new domestic cat genome assembly based on long sequence reads empowers feline genomic medicine and identifies a novel gene for dwarfism," PLoS Genetics 16(10): e1008926 on October 20, 2020, reported a revisit of the genomic sequence of Cinnamon, an Abyssinian breed domestic cat previously sequenced.  Rather than focusing on one cat, this group* performed whole genome sequencing (WGC) of 54 domestic cats and aligned the sequences to detect single nucleotide variants (SNVs) and structural variants (SVs).  The distribution and relatedness of the cats in this study is shown in this graphic:

Their aim was to identify the sequence comprising the ~300,000 gaps in the annotated sequence reported to the Cinnamon Abyssinian, to produce a new reference cat genome denoted in relevant databases as Felis_catus_9.0.  This genome comprised 2.84 gigabasepairs (Gb), of which only 1.8% (1.38 megabasepairs, Mb) was not assigned to a specific chromosomal location.  The sequencing and comparison identified 19,748 genes, 376 of which were novel (i.e., had not been identified in Cinnamon's DNA) and 178 genes found in the original annotations but not found here.  (The authors note the presence of 54 diverse individual cats from which the genes were determined as a possible source of this disparity.)  A greater extent of LINE/L1 repetitive elements were detected, which these researchers attributed to better assembly (i.e., the repetitiveness and gaps did not mask these elements in the latest assembly).  The average number of SNVs detected per cat was 9.6 million (for comparison humans have ~5 million); inbred cats showed a lower number (~8 million) while outbred cats showed a higher number (> 10.5 million).  Of these, researchers detected 128,844 synonymous SNVs in protein-coding sequences (i.e., where the two sequence encoded proteins having the same amino acid sequence), 77,662 missense SNVs (wherein the protein encoded differed in amino acid sequence, including truncated sequences and out-of-frame mutants), and 1,179 loss-of-function (LoF) SNVs.  LoF and missense SNVs were associated with depletion in comparisons between orthologous cat and human DNA sequences, whereas synonymous SNVs were enriched by 19.2%.  Statistical analyses found that in cats and humans there was selection against missense and LoF SNVs in genes under selective pressure (i.e., where the genetic changes resulted in phenotypes deleterious to individuals carrying them).  These results further indicated that these classes of SNVs were not distributed randomly in the cat or human genome.

Turning to structural variants, these researchers detected an average of 44,900 SVs per cat (a frequency four-fold higher than in humans), comprising 134.3 Mb; of the structural variant types, deletions averaged 905 bp, duplications 7,497 bp, insertions 30 bp, and inversions 10,993 bp.  In total they reported 208,135 SVs detected in 54 sequenced cat genomes, with 123,731 (60%) were deletions, approximately 39,955 insertions, 35,427 inversions, and 9,022 duplications were identified.  The majority of these were common across cat breeds, indicating tolerance. 58.15% of these SVs were intergenic in location, 40.22% intronic, and only 1.06% found in exons, "potentially impacting 217 different protein coding genes.  As the scientists noted:

Conversely, the proportion of some SV types found in certain gene regions varied from their genome-wide averages.  For example, in regions 5 kb upstream and downstream of genes, duplications were increased approximately two-fold.  For exonic regions, 74% of SVs were deletions, an increase from the genome wide level of 59.45%.  For 5' untranslated regions (UTRs), the majority of SVs were inversions, which only represent 17.02% of total SVs.  These results suggest an interaction between the impact of SV types and the potential function of the gene regions they are found in.

Sixteen of the identified variants were predicted to cause disease, based on comparisons with human genome sequences known to be sufficiently similar.  These included one involving a tumor suppressor gene FBXW7 associated with feline mediastinal lymphoma, prevalent in Siamese cats and Oriental shorthaired cats.  Other variants detected and their related diseases were in the FAM13B gene, resulting in ectodermal dysplasia (in a random-bred cat); the CYFIP2 gene associated with urate stones (Egyptian mau); and the SH3PXD2A gene, associated with feline infectious peritonitis (random bred cat).

The genetic roots of dwarfish more complicated according to these researchers, involving "a complex deletion coupled with a nearby potential duplication event" on cat chromosome B1, that disrupted the gene for UDP-glucose-6-dehydrogenase (UGDH).  This trait is inherited as an autosomal dominant mutant and a breed-standard for Munchkins, characterized by shortened limbs and anormal torso.  Here, the scientists report that:

SV analysis within the critical region previously identified by linkage and GWAS on chromosome B1:170,786,914–175,975,857 revealed a 3.3 kb deletion at position chrB1:174,882,897–174,886,198, overlapping the final exon of UDP-glucose 6-dehydrogenase (UGDH).  Upon manual inspection of this SV, a 49 bp segment from exon 8 appeared to be duplicated and inserted 3.5 kb downstream, replacing the deleted sequence.  This potentially duplicated segment was flanked by a 37 bp sequence at the 5`end and a 20 bp sequence at the 3' end, both of unknown origin.

(This topography is illustrated in the Figure below.)  The scientists further hypothesized that the known activity of the affected gene, UGDH, which is involved in "proteoglycan synthesis in chondrocytes" could effect this phenotype by interfering with development.  Evidence for this mechanism was observed in the epiphyseal plate, which in Munchkins showed "a disorganized columnar arrangement" and proteoglycan depletion.  They report that sequencing the mRNA produced by the mutant UGDH is yet to be reported, which would perhaps shed light on whether the role of this mutation was as they propose it to be.

This group published a more recent paper in March 2021, entitled "Ultracontinuous Single Haplotype Genome Assemblies for the Domestic Cat (Felis catus) and Asian Leopard Cat (Prionailurus bengalensis)," in the Journal of Heredity, 112(2): 165–73, providing additional details relating to its analysis of the domestic cat and leopard cat components of the hybrid genome.

The results reported in these papers illustrated how far we have come and perhaps how far we have to go in using genetic methods for identifying the causes and possible cures for a variety of diseases.

*Reuben M. Buckley, Brian W. Davis, Wesley A. Brashear, Fabiana H. G. Farias, Kei Kuroki, Tina Graves, LaDeana W. Hillier, Milinn Kremitzki, Gang Li, Rondo P. Middleton, Patrick Minx, Chad Tomlinson, Leslie A. Lyons, William J. Murphy, Wesley C. Warren

Nautilus pompilius Genome Determined

By Kevin E. Noonan --

Cephalopods are fascinating creatures, and their primary living representative -- the octopus -- has recently been the subject of the Academy Award winning documentary "My Octopus Teacher."  They are clearly intelligent (as set forth in Peter Godfrey-Smith's Other Minds: The Octopus, the Sea, and the Deep Origins of Consciousness), but it is an intelligence so different from human intelligence that it was an inspiration for the alien first-contact film, Arrival.

Outside the coleoid group, the only living relative are animals of the genus Nautilus, the sole surviving externally shelled cephalopod since the Paleozoic (541 to 252 million years ago).  That shell is itself iconic, as both objet d'art and a living illustration of the Fibonacci series in the ratios of the whorls from which the shell is made.

Recently, an international group of researchers (predominantly working in China)* published a paper entitled "The genome of Nautilus pompilius illuminates eye evolution and biomineralization," in the July edition of Nature Ecology & Evolution, elucidating the Nautilus genome.  (The octopus genome has been determined previously; see "Octopus Genome Sequenced".)  In stark contrast with the octopus genome, the Nautilus genome is parsimonious, the authors characterizing it as "compact, minimalist" with few genes and "slow evolutionary rates in both non-coding and coding regions."  Genetic innovations in Nautilus involve gene loss, with expansion and contraction independently of certain gene families resulting in the "pinhole" eye which functions without either cornea or lens.  These distinctions with their coleoid cephalopod cousins provide the possibility that genomic comparisons could provide insights into how evolution shaped both cephaloid types, according to the authors.

These researchers report that the Nautilus genome comprises 731 megabasepairs, which is the smallest of the cephalopods, being 2.5-7 fold smaller than their extant coleoid relatives.  One feature the authors called "strikingly different" in Nautilus DNA is found in transposable elements (TE), known to be "the driving force in shaping genomic architecture and evolution."  TEs comprise 31% of the genome, with retrotransposons, including LINE (long interspersed nuclear element) SINE (short interspersed nuclear element), and LTR (long terminal repeats) elements constituting only a minor portion (6.5%); in contrast, these elements are "a prominent presence" in the coleoid cephalopod genome.  Moreover, further analysis indicated that an ancient DNA transposon "burst" occurred once in the Nautilus lineage and that there has been no further expansion of TEs in the Nautilus genome (coleoid cephalopods have had more evolutionarily recent instances of such bursts).  Nautilus shares this characteristic with other mollusks, which the authors say suggest slow evolutionary rates in non-coding regions of the Nautilus genome.

Genetic analyses identified 17,710 protein-coding sequences in the Nautilus genome, which is only 53-61% of the number of putative gene sequences found in octopus and squid genomes.  On the gene family level this is reflected by "a huge contraction of orthologous gene families" in the Nautilus genome (204 gene families contracted versus only 9 gene families that have expanded).  The authors note that 18 domains of centromere protein B were specifically expanded in Nautilus compared with other cephalopods; this protein is thought to "play[] crucial roles in host genome integrity and replication fidelity through the repression of retrotransposons and centromere formation in yeast or humans" consistent with the frequencies of TE sequences found in the Nautilus genome.

On a more global level, comparisons of the Nautilus genome to related (and not-so-related) species was used to assess phylogenetic relationships, using 423 single-copy orthologues from 16 animal genomes as shown in the figure:

Divergence from the coleoid cephalopods was estimated by these studies to have occurred around the Silurian-Devonian boundary at about 423 million years ago (Ma), consistent with other estimates from geological evidence and "molecular clock" inferences.  On a finer scale, these researchers report that N. pompilius populations expanded about 23 Ma ("at the turn of the Miocene") in a "stepwise manner," which expansion then stopped as a result of climate changes (glaciation) at the Mid-Pleistocene Transition (around 1 Ma), and finally fell precipitously around 0.4 Ma during another intense glaciation period termed the Mid-Brunhes Event.  These authors conclude that this pattern indicates N. pompilius to be sensitive to "extreme environmental fluctuations" (although it is not evident that they are particularly more sensitive than other aquatic species).  Paradoxically octopus species and certain bony fishes thrived during this period according to the paper, suggesting "ecological competition" (i.e.,"nature red in tooth and claw") had much to do with the population record of N. pompilius during these times.

Turning to the pinhole eye, these researchers report the presence of "nearly all" of the genes encoding the core regulatory complex of transcription factors thought to govern lens formation in coleoid cephalopods (PAX6, SIX3/6 and SOX2, among others) in Nautilus, consistent with lens development in the early Cambrian era (541-485 Ma).  In Nautilus, an important regulatory gene for lens production (in vertebrates as well as coleoid cephalopods), Nrl/Maf, is missing and genes encoding lens-specific crystallin genes are "contracted" compared to other mollusks like octopus that have lens-equipped eyes.  Other specific crystallin genes found expanded in coleoids are missing in the Nautilus genome.  Nautilus is known for behaviors (such as coming towards the surface to feed under lower light conditions) indicating visual perception, and were found by these researchers to possess one photoreceptive r-opsin gene and one retinochrome gene, which these authors assert represents "the minimal opsin gene number among known metazoans" (albeit conceding that N. pompilius is almost certainly colorblind).  The authors note that light intensity is a more critical faculty for animals like N. pompilius that migrate vertically between depths at sea, and that such sensitivity depends on the 11-cis retinal chromophore which isomerizes in response to light.  The N. pompilius genome contains a retinochrome encoding such an isomerase, and in addition these researchers identified an ortholog of a gene family (ten genes) named retinal pigment epithelium-specific protein 65 kDa present in vertebrates that is also found in the Nautilus genome.  These genes contain a conserved iron binding site, an "active cavity" site, and a "hydrophobic tunnel" consistent with catalytic activity.  The significance of these finding is explained by the authors as:

From a perspective of evolutionary adaptation, the appearance of the pinhole eye is one adaptive breakthrough essential to the nautilus lifestyle of vertical depth migrations, allowing the organism to acquire spatial vision and rapidly cope with hydrostatic pressure within the eye through opening the pupil to seawater.  Overall, multiple genomic innovations including gene losses, independent contraction and expansion of specific gene families and presence of associated regulatory networks seem to work in unison to drive the evolution of the pinhole eye in nautilus.

The paper provides a schematic diagram of the Nautilus pinhole eye to put these genetic analyses in context:

Turning to the other characteristic morphological feature of Nautilus, its shell, the authors note that it is made up of aragonite (an orthorhombic crystal of calcium carbonate, CaCO₃) and shell matrix proteins (SMP) used as a substrate.  The authors report finding genes encoding a total of 78 SMPs with expression pattern concentrated in the shell mantle (72%).  Genetic comparisons revealed that 21 Nautilus SMPs shared sequence similarity with analogous genes from bivalves and gastropods and found several conserved domains in these protein across mollusk species, including "laminin, chitin-binding and carbonic anhydrase domains."  The authors posit these regions as part of a "core biomineralization toolkit" conserved across external shell-expressing mollusks.  On the other hand, 52 of the 78 SMPs encoded in the Nautilus genome were either new or Nautilus-specific, and these researchers speculate that most of the unique SMPs evolved independently in different mollusk species including Nautilus.  Indeed, there is one important SMP, Nautilin-63, that these researchers found showed low sequence similarity even within the Nautilus genus.  And the top ten SMPs enriched in the Nautilus mantle contain an evolutionarily novel poly(Gly or Gly-Ala) repetitive motif; also detected encoded in these proteins were several repetitive low-complexity domains (RLCDs), in common with other shelled mollusks, that the inventors suggest "could be a unifying principle for molluscan biomineralization, especially for nacre formation."

Finally, the paper discusses the Nautilus immune system, which contains a "highly complex yet comprehensive innate immune components" enabling Toll-like receptor and tumor necrosis factor receptor signaling that regulates "apoptosis, inflammation and immune defences."  Also detected were genes encoding IL17R, H-lectin, and IL1, which the authors assert "supports the assumption that nautilus has preserved a more complete repertoire of immune molecules than other cephalopods."  An assessment of gene numbers for immune system-specific genes showed expansion of C-type lectin genes (81) compared to coleoid species (wherein 12-33 genes are encoded) and some of these genes have been independently duplicated.  Also found to be specifically expanded are interferon-inducible GTPases, genes also known to be implicated in immune function.  These genetic features of the immune system-involved genes in Nautilus (and comparisons with coleoid species) is illustrated in this figure:

The authors conclude with this synopsis of their findings:

Genomic evidence reveals that nautilus has undergone lineage-specific innovations in both body plan and behaviour since the Cambrian and retained these extraordinary features after a long evolutionary history.  In particular, vertical depth migration in Nautilus and other chambered cephalopods is one of several critical and common strategies needed to avoid predators and budget energy; these may have helped the survival of these species ever since.  The emergence of the pinhole eye is a great innovation for switching from directional to spatial vision and rapidly change hydrostatic pressure, making vertical depth migration possible.

Our findings highlight that co-evolutionary loss of core regulatory transcription factors may have driven the evolution of the pinhole eye.  Moreover, our proteomic and transcriptomic data suggest that an ancient "core biomineralization toolkit" and new RLCDs coordinately directed the construction of the chamber shell, which has evolved into the buoyancy apparatus needed to adapt to a critical life mode.  Taken together, the draft genome of N. pompilius together with multiomics provide a valuable insight into not only the adaptive innovations of the ancestor of cephalopods but also the dynamic evolution of coleoids.

* Key Laboratory of Tropical Marine Bio-resources and Ecology and Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou; Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou; Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou; MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale, CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei; Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo; Biomarker Technologies Corporation, Beijing; State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou; Department of Neuroscience and Developmental Biology, University of Vienna.

Great Dane Genome Reveals Involvement of Retrotransposition in Canine Diversity

By Kevin E. Noonan --

The domestic dog (Canis lupus familiaris) has been the subject of many genetic studies, particularly since the dawning of the age of Whole Genome Sequencing in the late 20th Century.  These studies have elucidated some interesting facts about "man's best friend," some of which have been discussed here (see "The Genetic Basis of Coat Variation in Dogs"; "Leg Length Variation in Dogs and its Relevance to Human Mutations"; "From Toy Poodle to Rottweiler: Why Is Fido So Small (or Large)?";  "Selection for Facial Features in Domestic Dogs: The Evolution of Cuteness") and comparators to related species (see "Red Fox Genome Sheds Light on Domesticated Dogs (and Maybe Humans)").

But as with other organisms, a great deal of the genetic sequence set forth early in the 21st Century was incomplete due to technical limitations. Chief amongst these was difficulty in obtaining reliable sequence information on sequences repeated in many places in the genome, which interfered with reliable assembly of sequenced portions (termed "contigs") into longer (ideally, chromosomal-length) linear sequence assemblies (termed "scaffolds).  Improvements in sequencing technology now permit better sequence determinations for high GC and highly repetitive regions.

The existence of a prior reference genome (produced from a female boxer named Tasha), combined with these new sequencing tools, was used by an international team of researchers* in a paper published in March of this year in the Proceedings of the National Academy of Sciences (USA) entitled "Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes."  These scientists reported elucidation of the genomic sequence from a female Great Dane named Zoey, wherein they were able to identify and eliminate (in large part) incorporation of false contigs due to repetitive ends and fragment ends that map to multimer genomic locations.  Using alignment with the reference dog genome revealed gaps between contigs as earlier reported in dog genomic DNA.  They report finding 373 additional sequence regions spanning contigs having N50 of 30kb and a total length of 10.5 Mbp.  The resulting aligned scaffolds were assigned to dog chromosomes to constitute a genomic sequence for the dog genomic complement of 78 chromosomes.

From this sequence the researchers were able to identify 22,182 protein-coding gene models; full-length matches with the reference canine genome were found for only 84.9% (18,834) of all protein-coding gene models, and almost full-length alignments were found for 93% (20,670) of the models; in addition they identified 49 protein-coding genes not present in the earlier canine reference genome.  Also annotated were 7,049 long noncoding RNAs that included 84 with no or only partial alignment to the reference canine genome.  From this comparison they further were able to appreciate that the assembly from the Great Dane genome spanned the majority of sequence gaps not having been sequenced in the reference dog genome.  Mapping of these gaps showed that 2,151 gaps (16.8% of gaps) overlapped the transcription start site of a predicted protein-coding gene and were found to be extremely GC rich (67.3%).  Further, a subset of the resolved gaps, having a median 80.95% GC content, were found to be preferentially localized (i.e., at a frequency greater than random chance) at transcription start sites and recombination hotspots.  About 12% (1,457 of 12,304 on the autosomes) of gap segments were found to be located within 1 kbp of a hotspot (the expected percentage of such location was closer to 3%).  These hotspot-adjacent segments were extremely GC rich; 5,553 such identified segments had a GC content greater than that what would be expected from random sequence permutations.  The total extent of these extreme GC segments spanned 4.03 Mbp in the Great Dane sequence assembly, were found to have "a median length of 531 bp, a median GC content of 80.95%, and are located much closer to transcription start sites (median distance of 290 bp) and recombination hotspots (median distance of 68.7 kbp) than expected by chance."

Analysis of the Great Dane genome assembly with the reference canine genome identified 16,834 deletions (median size: 207 bp) and 15,621 insertions (median size: 204 bp) in Great Dane DNA.  Genetic assessment of these sequences revealed predominantly the presence of two forms of "retrotransposon insertion/deletion polymorphisms" which included dimorphic canine short interspersed elements (SINECs) (16,221 copies) and dimorphic long interspersed element-1 sequences (LINE-1_Cfs) (1,121 copies).  The 3' flanking sequence for the LINE-_Cfs elements suggested "multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations."

The researchers further reported a length distribution of the detected variants having "a striking bimodal pattern, with clear peaks at ∼200 bp and ∼6 kbp, consistent with the size of SINEC and LINE-1 sequences," as shown in this Figure:

The location of these sequences was associated with insertions and deletions, the researchers reporting that inspection of the sequences in the 150-250-bp range (dimorphic SINEC elements) were found at 7,298 deletion and 6,071 insertion sites, and that "LINE-1 sequences accounted for 339 deletions and 581 insertions longer than 1 kbp." When combined with data from the reference canine genome there were at least 16,221 dimorphic SINEC and 1,121 dimorphic LINE-1 sequences identified.  "Hallmarks of retrotransposition" were identified at these sites, wherein the SINEC and LINE-1 sequences were flanked by "target site duplications having a 15 bp median size with the elements ending in poly(A) tracts having median lengths of 9 bp to 12 bp."

To test whether these sequences were capable of retrotransposition, a particular sequence was cloned that had intact open reading frames encoding the ORF1p and ORF2p predicted proteins and lacked mutations expected to disrupt protein function.  This element was introduced into human cells in vitro and shown to be capable of retrotransposition, as illustrated in this Figure:

This element also capable of mobilizing both the canine SINEC elements and analogous human Alu elements.  The researchers speculated that this result was consistent with ongoing retrotransposon activity as a driver of canine genetic variation.

The researchers set forth a synopsis of their results as follows.  They had identified 49 predicted protein-coding genes from the Great Dane assembly that were not found in the canine genome, as well as 2,151 protein-coding gene models having a transcription start position located in a gap in the reference genome sequence.  The existence of high GC-content sequences in canine promoter regions distinguishes the preferential location of recombination events in dogs, which lack a functional PRDM9 gene known to mediate recombination in other mammals; see Paigen & Petkov, 2018, "PRDM9 and its role in genetic recombination," Trends Genet. 34, 291–300, and Auton et al., 2013, "Genetic recombination is targeted towards gene promoter regions in dogs," PLoS Genet. 9, e1003984.  The researchers assert that "[t]he presence of extremely GC-rich segments likely reflects a key aspect of canine genome biology" as a consequence of these findings.  Further, a comparison of the number of single nucleotide variants (i.e., differences) found in Zoey and Tasha (3.57 million) was lower than found in similar comparison between humans (4.1-5.0 million).  In contrast, the levels of LINE-1 and SINEC dimorphism between these two dog genomes was "disproportionately large," there being "an ∼17-fold increase in SINE differences (16,221/915) and an eightfold increase in LINE differences (1,121/128) compared to the numbers found among humans" (indeed, the researchers report that "more dimorphic SINEs were found between these two breed dogs than have been found in studies of thousands of humans").  They note that these results are consistent with prior studies, including Wang & Kirkness, 2005, "Short interspersed elements (SINEs) are a major source of canine genomic diversity," Genome Res. 15, 1798–808.

The authors conclude by saying:

[O]ur study suggests that retrotransposition is an ongoing process that continues to affect the canine genome.  We provide proof-of-principle evidence that dog genomes contain LINE-1 and SINEC elements that are capable of retrotransposition in a cultured cell assay.  We also identified two LINE-1 lineages with the same 3′ transduced sequence associated with multiple elements, suggesting the presence of multiple canine LINE-1s that are capable of spawning new insertions.  Additionally, analysis of 3′ transduction patterns suggests the presence of additional active LINE-1s in canines that have yet to be characterized.  Thus, a full understanding of canine evolution and phenotypic differences requires consideration of these important drivers of genome diversity.

* From  the Department of Biological Sciences, Bowling Green State University; the Department of Human Genetics, Department of Computational Medicine and Bioinformatics, and the Department of Internal Medicine, University of Michigan; the Université Côte d'Azur, CNRS, INSERM, Institut de Recherche sur le Cancer et le Vieillissement de Nice, Nice; the Université de Rennes 1, CNRS, Institut de Génétique et Développement de Rennes−UMR 6290, Rennes; and the Department of Biomedical Sciences, Cornell University, Ithaca, NY 14850.

BIO Declaration on Global Access to COVID-19 Vaccines and Treatments and Role of IP

By Donald Zuhn --

In a message distributed to Biotechnology Innovation Organization (BIO) members, Dr. Michelle McMurry-Heath, BIO President and CEO, released a declaration signed by the corporate and organizational leaders of 209 global biotechnology companies and 41 biotech associations, in which the signatories acknowledged their "social responsibility to work with other stakeholders -- healthcare providers, governments, multilateral organizations, and non-governmental donor organizations -- to ensure that COVID vaccines and treatments get to the patients in the world who most need them."

Dr. McMurry-Heath began her message to BIO members by recognizing "the ongoing effort by some political leaders to strip our scientific intellectual property rights pertaining to vaccines and treatments for COVID-19," and noting that "[t]his proprietary science and technology was developed by our scientists working day and night at great financial risk to our companies."  The BIO CEO also highlighted BIO's efforts to establish a COVID-19 global Strategy for Harnessing Access Reaching Everyone (SHARE) program, which seeks to ensure a sufficient global supply of COVID-19 vaccines, to ensure safe and expeditious global access to COVID-19 vaccines and therapeutics, and to strengthen and support healthcare systems in low-and middle-income countries in addressing COVID-19.  Dr. McMurry-Heath also pointed out that biotech companies "ARE sharing our IP and know-how through almost 300 voluntary global agreements to increase vaccine manufacturing and distribution capacity."  Yet, the BIO CEO notes that these efforts have "not changed the minds of those who believe we should simply turn over all of our science to countries around the world," an idea Dr. McMurry-Heath asserts "is misguided, and most importantly, won't solve the problem."

The declaration sets forth five statements or goals.  First, the signatories state that the biotech sector "must continue to play a constructive, proactive part in developing COVID solutions and the global manufacturing capacity to produce them."  The declaration notes that in the past year, more than 950 global R&D projects have been launched on COVID-19 vaccines, treatments, and biologics, and more than 250 global partnerships have been formed to build manufacturing capacity.

The declaration next states that intellectual property is the foundation of the biotech sector, explaining that:

[Intellectual property] is responsible for creating the global biotech network that responded so quickly to the COVID crisis in the first place.  It is what gives investors the confidence to fund companies with long time horizons and high risks.  It gave companies the assurance that they could quickly pivot during the early days of the pandemic into COVID projects.  And it helped ensure the type of global cooperation and partnerships that are driving companies, countries, and manufacturers to quickly scale up the production.

The signatories also express their support for strong, collaborative efforts like those endorsed by the G-20 to address the global imbalances in access to COVID vaccines and treatments.

Turning to the proposed World Trade Organization (WTO) waiver of intellectual property rights, the signatories declare that the proposed waiver "will be ineffective and counterproductive in addressing this crisis," arguing that:

Intellectual property rights are not responsible for the imbalance in COVID vaccine supplies between higher and lower income countries.  It will create a long contentious global negotiation that will not urgently address the crisis, and foster more "vaccine nationalism," exacerbating shortages in an already strained global supply chain.  It would divert limited resources from companies that are focused on maximizing current global partnerships while maintaining quality and patient safety.  Lastly, it would send a powerful signal to the biotech sector and investors to avoid taking the risks to develop solutions in future public health emergencies.

The declaration concludes with the goal of producing more than 11 billion doses of COVID-19 vaccines in 2021, and significantly more in the first part of 2022, and a commitment by the signatories to work with other global stakeholders "to see that these doses get to those that most need them, wherever they may be."

A list of the corporate and organizational signatories to the declaration can be found here.

Science Does Not Support the Latest COVID Hysteria

By Kevin E. Noonan --

In a time of a global pandemic, with antivaxxer and anti-science sentiments running rife, and when combinations of fear, distrust, and paranoia are rampant, it is easy for important results from basic science to become fodder inaccurately supporting the doomsday memes that abound.  This is the story of a scientific paper from two eminent scientists that, properly understood, explains a puzzling aspect of SARS-CoV-2 biology in COVID-19 patients:  why can viral genetic information be detected in some patients months after they have recovered from their illness, using sensitive testing (polymerase chain reaction, or PCR; for reference see CSI).  As it turns out, it is not evidence that SARS-CoV-2 is manmade, or that the various vaccines against the disease are toxic or disease-causing, or that (as Senator Rand Paul (R-TN) is (in)famous for charging) Dr. Anthony Fauci supported "gain-of-function" research in Wuhan China and then tried to cover it up.  There are plenty of websites having that content; here we will strive to stick to the facts.

The paper, "Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues," appeared in the Proceedings of the National Academy of Sciences USA this week, and was authored by Rudolf Jaenisch and Richard Young and their colleagues.  The purpose of the paper as stated therein is to resolve "[a]n issue of SARS-CoV-2 disease is that patients often remain positive for viral RNA as detected by PCR many weeks after the initial infection in the absence of evidence for viral replication," i.e., in the absence of producing live virus.  Their hypothesis:  perhaps enzymes produced in human cells, capable of converting RNA (like messenger RNA) to DNA, was introducing some portions of the SARS-CoV-2 RNA into the genome of infected cells.  This RNA would then be detected after the RNA was made as part of gene sequences surrounding the integration site.

It should be appreciated in this regard that a great deal of human DNA, colloquially termed "junk" DNA, does not encode proteins or regulatory sequences (which is the function of the remainder of genomic DNA and was what earlier workers believed to be the totality of the genome).  This seemingly non-functional DNA is made up in significant part by the remnants of ancient retroviruses and retrotransposons, DNA elements "reverse transcribed" from RNA to DNA and inserted in the genome (perhaps randomly).  Some of these species form so-called repetitive elements; one of the most prevalent in human DNA (17%) named Long Interspersed Nuclear Element 1 (LINE1):

These repeats, like their retrovirus and retrotransposon ancestors, can express a form of reverse transcriptase that can act on cellular RNA and result in reinsertion of the resulting DNA into the cellular chromosome (this is not an unknown phenomenon and is not necessarily associated with virus infection when it has been detected; see, Horie & Tomonaga, 2011, "Non-retroviral fossils in vertebrate genomes," Viruses 3, 1836–1848).  Importantly to this work, other RNA viruses and even cellular genes have been known to have portions of their RNA retroinserted into cellular genomic DNA after reverse transcription by endogenous cellularly encoded reverse transcriptases.

Drs. Jaenisch and Moore first studied whether SARS-CoV-2 RNA could be reverse transcribed and reinserted into the genomic DNA from cultured human cells in a Petri dish; the cells they used are an established, immortal cell line known as HEK293T.  These cells arose from human embryonic kidney and differ from normal cells by not experiencing aging, senescence, and cell death after a limited lifespan.  They also contain DNA from a transforming adenovirus, having been made in 1973 and continuously grown (or "passaged") since then.  Their chromosomal complement is abnormal, having on average 64 chromosomes and multiple copies of some of them (e.g., 3 copies of the X chromosome and 4 copies of chromosomes 17 and 22).  In short, these are not normal cells but nevertheless have proven to be very useful for a variety of purposes in cell biology studies and biotechnology.

In the COVID-19 studies reported in this recent paper, these scientists increased the likelihood of observing what they expected to be extremely rare events by introducing into recipient HEK293T cells a cloned copy of human LINE1 genetically engineered to express the endogenous reverse transcriptase (it will be appreciated that such cells do not occur naturally).  When assayed, using PCR detection or by genomic DNA sequencing methods, the largest SARS-CoV-2 derived DNA sequence was 1,632 basepairs (bp) encoding the protein (the nucleocapsid, NC) that acts as a "shell" for the viral RNA, in a genetic milieu that validated the hypothesis for LINE1-mediated reverse transcription and integration into the genome.  These studies also showed that portions of human genes called exons were preferential (76%) targets for integration, providing an explanation for why PCR treatment of cellular RNA could detect portions of SARS-CoV-2 RNA in otherwise virus-free individuals post-infection.  Similar results at lower frequencies were detected in HEK293T cells not having artificial LINE1-associated reverse transcriptase encoded by a genetically engineered LINE1 element.

When the presence of chimeric human/SARS-CoV-2 RNAs were assessed in samples from infected individuals they were found at very low frequency -- 0.004–0.14% of the total detected virus-related species, although they were found in multiple tissue types including lung, heart, and brain tissues.  Most of these detected sequences were from NC-encoding RNA, consistent with the HEK293T cell experiments.  That the SARS-CoV-2 RNA detected in human samples had been produced from integrated fragments of this RNA was supported by further findings that about 50% of these sample fragments had been produced by "negative-strand" (i.e., the complement in a double-stranded DNA) species that could only have come from such DNA (because the virus RNA is "positive stranded," i.e. the strand that contains the transcript in a form that can be directly translated into protein in the cell).

These results explain the scientific conundrum of finding evidence of SARS-CoV-2 RNAs in individuals who had recovered from COVID-19 and showed neither symptoms nor the presence of SARS-CoV-2 virus in clinical samples.  It also suggested that continued expression of SARS-CoV-2 chimeric RNAs (and the proteins encoded thereby) might result in a persistence of immunity because the individual's immune system would continue to recognize them as antigenic and produce an immune response against the virus in the absence of later re-infection.

These results show the ability of scientific inquiry to resolve unknown and unsuspected aspects of SARS-CoV-2 biology, unsurprising for a virus only recently identified and (if current etiological explanations hold) only recently having humans as a host (see "Evolution of SARS-CoV-2 from Bat to Human Pathogen").  But it is clear that what these results do not support is a conclusion that the mRNA vaccines (from Pfizer/BioNTech or Moderna) are in any way unsafe; after all, besides being the consequence of natural processes existing through geological time the exceedingly small frequency of these events is inconsistent with realistic worries about vaccination (considering the much more severe and frequent alternative outcome for those infected with the virus).  The advent of the first pandemic in over a century,  particularly coming after a little more than a century of medical and scientific achievements that "conquered" infectious diseases, is disquieting if not frightening.  But it is a mistake to let that fear inhibit use of the product of over a century of medical and scientific inquiry and successful intervention against infectious disease.  As Dan Rather was known to say, "Courage."

Top Drugs Losing Patent Exclusivity in 2021

By Kevin E. Noonan --

Earlier this month, Eric Sagonowsky reviewed the top ten drugs in the U.S. (in terms of sales) losing patent exclusivity in an article published by Fierce Pharma.

These drugs are Lucentis (Genentech/Roche), Bystolic (AbbVie/Allergan), Vascepa (Amarin), Nothera (Lundbeck), Narcan (Emergent Biosolutions), Brovana (Sunovion), Sutent (Pfizer), Saphris (AbbVie/Allergan), Amitiza (Mallinckrodt), and Feraheme (Amag Pharma).  The nature of these losses and consequences thereof can be seen from the article, synopsized here.

Lucentis (ranibizumab), Roche's drug for macular degeneration, is a humanized mouse monoclonal antibody fragment specific for vascular endothelial growth factor A.  It is related to Roche's Avastin (bevacizumab) product, having been modified for injection into the vitreous humor of the eye for the treatment of wet age-related forms of the disease (AMD).

Bystolic (nebivolol), AbbVie/Allergan's drug for high blood pressure, is a small molecule beta blocker having the formula:

The last patent on this drug product expired on December 17, 2021; under the terms of settlement agreements with several generic competitors, versions of the drug can come on the market (if FDA approved) on September 17, 2021.  However, antitrust litigation by direct purchasers may complicate this scenario.

Vascepa (ethyl eicosapentaenoic acid), Amarin's drug for hypertriglyceridemia and cardiovascular disease, is a purified omega-3 fatty acid eicosapentaenoic acid having the formula:

The drug is involved in litigation, Amarin having lost its ANDA litigation action against Hikma Pharmaceuticals (see "Amarin Pharma, Inc. v. Hikma Pharmaceuticals USA Inc. (Fed. Cir. 2020)"), although Amarin brought another lawsuit against Hikma in November 2020.

Nothera (droxidopa), Lundbeck's drug for neurogenic orthostatic hypotension, is a synthetic amino acid analogue that is converted in vivo to norepinephrine and has the formula:

Several generic challengers are in the offing, including Camber Pharma (which launched its generic in February 2021), Hikma (March 2, 2021), and Zydus Cadila (FDA approval May 2020).

Narcan (naloxone) is Emergent Biosolutions's drug known for treating opioid overdose and has the formula:

This drug has been in ANDA litigation with Teva, which received FDA approval in 2019 and invalidated Emergent's Orange Book-listed patents in the district court; that decision is on appeal to the Federal Circuit.  Emergent reached a settlement agreement with Perrigo that licenses the company to bring its generic Narcan to market in 2033 or earlier should Emergent be unable to obtain reversal of the district court's decision invalidating its patents.

Brovana (arformoterol) is Sunovion's drug for treating chronic obstructive pulmonary disease (COPD).  It is a long-acting β₂ adrenoceptor agonist having the formula:

All Sunovion's patents protecting this drug will expire in 2021, and several generic companies have received tentative FDA approval.

Sutent (sunitinib), is marketed by Pfizer for the treatment of cancer including gastrointestinal stromal tumors, advanced renal cell carcinoma, and pancreatic neuroendocrine tumors.  It has the formula:

All the drug's patents expire in 2021, and generic competitors include Glenmark and Mylan (which lost an ANDA litigation over its proposed generic version of the drug in 2010).

Saphris (asenapine) is AbbVie/Allergan's drug for treating schizophrenia and bipolar disorder.  It has the formula:

Breckenridge, Alembic, and Sigmapharm launched generic versions of the drug in December 2020.

Amitiza (lubiprostone) is Mallinckrodt's drug for treating constipation and irritable bowel syndrome.  It has the formula:

Mallinckrodt recently filed for Chapter 11 bankruptcy.

Feraheme (ferumoxytol) is an intravenously administered, carbohydrate-coated, superparamagnetic iron oxide (Fe₃O₄) nanoparticle inorganic product for treating anemia marketed by Amag Pharmaceuticals.  Under the terms of a patent infringement litigation settlement, Sandoz is authorized to launch its generic version in July 2021 (having received FDA approval in January 2021).  Other provisions of this settlement provide for Sandoz to pay royalties to Amag until June 30, 2023 (when the last patent expires) and for Amag to market an authorized generic in July 2022.

The economic consequences for these companies can be assessed by their U.S. sales, although as Mr. Saganosky reports, not all of them will be facing generic or biosimilar competition right away (for various reasons including court battles and regulatory approval delays).

* 2019 U.S. sales data

Mr. Sagonowsky notes that his report is based on information "from numerous sources, including lists of potential generic launches from OptumRx, GoodRx, GreyB, and Corporate Pharmacy Services, plus company filings, conference calls with analysts, FDA records and more."

Peach Genome Dissected to Provide Insights into Ecological Influences

By Kevin E. Noonan --

The humble peach has been the inspiration for pies, album titles, independent movies, and a fictional woman's baseball team, but is also an important food species, yielding 24.5 million tons globally in 2018.  Like all plant species, their sessile nature makes them a good target for environmental studies because, unlike most animals, they must respond to changes in their environment by adaptation rather than flight.  They have a diverse temperate host range from their origins in southwestern China, being found in temperate and subtropical regions, wet and dry climates, and in harsh environments (high altitudes, severe cold, and drought conditions).  This range suggests that the peach is a good species for studying climate effects on their evolution as illustrated by genetic changes, particularly in view of the climate change the globe has been experiencing for the past ~40 years that is expected to continue.

An international group of researchers* recently reported the results of their taking this suggestion, in a paper entitled "Genomic analyses provide insights into peach local adaptation and responses to climate change" in the journal Genome Research.  The basis for the study was 263 diverse accessions that consisted of 52 wild relatives and 211 landraces ("a domesticated, locally adapted, traditional variety of a species of animal or plant that has developed over time, through adaptation to its natural and cultural environment of agriculture and pastoralism, and due to isolation from other populations of the species").  The 263 peach accessions could be divided into seven major groups:  YG (Yun-gui plateau), NW (Northwest China), NP (North Plain China), YT (Yangtze River Middle and Backward), NE (Northeast China), TB (Tibet plateau), and ST (South China Subtropical) groups.  Placing their genome assay results in context, these researchers reported on the effects of glaciation, which had caused a sharp decline of effective population size during the two largest Pleistocene glaciations:  the Xixiabangma glaciation (that occurred 1.17–0.8 million years ago, mya) and the Naynayxungla glaciation (occurring 0.78–0.50 mya); there was also a smaller decline in population size during the "last glacial maximum" (LGM) about 20,000 years ago.  Also reported was a slight expansion of peach populations from the Tibetan plateau (TB) after the Naynayxungla glaciation about 300,000 years ago.

In total, 342.7 Gb of paired-end sequences were assayed, with a median depth of 5.3-fold corresponding to 91.7% of a reference peach genome ("Lovell"); the peach genome is relatively small (227.4 Mb) compared with other fruit species (e.g., apple, 650 Mb; and strawberry, 805 Mb).  These sequencing studies resulted in the identification of 4,611,842 single-nucleotide polymorphisms (SNPs), with 1,931,310 residing in introns (∼11.33%) and another 848,638 (∼4.98%) in protein-coding exons.  These researches also identified 1,049,266 small insertions and deletions (indels) (< 6 bp) and 106,388 larger structural variations (>30 bp).  These variations were used as genetic markers for mapping through time genomic changes and crossbreeding between the different geographically defined groups.  The results of these genetic analyses showed a pattern of extensive admixture and possible gene flow among landrace groups.

Genetic analyses identified specific loci involved in adaptation to local conditions.  These include a total of 2092 genomic regions found for all seven groups (comprising 19.1 Mb, ∼8.4%) and "189, 387, 301, 235, 280, 339, and 378 regions for the YG, NW, NP, YT, NE, TB, and ST groups, respectively."  These loci were termed candidate selection regions (CSRs) by these researchers, and together harbored 3742 genes, corresponding to 396, 966, 635, 403, 573, 743, and 680 genes for the YG, NW, NP, YT, NE, TB, and ST groups.  In addition, some of these genes were shared among different groups, "suggesting the unique adaptive patterns for each group and that different climates may shape distinct genomic regions" according to the authors.  Assessment of these genes identified several related to response to different types of stimuli and stress that were overrepresented; included were temperature, radiation, salt, DNA damage, osmotic, toxin, and biotic stimulus.  These results suggested to these researchers that these genes were involved in adaptive evolution in response to these stresses.

Turning to specific forms of environmental stress, the researchers found that peach specimens from high-salt soils in northwestern China were enriched for two cation/H+ exchanger family genes.  Additional genes found from to be enriched in these specimens were genes encoding proteins having a leucine-rich repeat (LRR) domain, comprising 121 of 612 members (∼19.8%).  These researchers noted that such genes were "considered to be one of the most important domains involved in plant resistance."  Pattern recognition receptor (PPR) proteins, which "form one of the largest protein families in land plants that are related to environmental responses" and are present with 286 members in the peach genome, were found in 79 (∼27.6%) of the identified candidate selection regions (CSRs).

Specific genetic characteristics were reported for certain groups.  For the YG group (which is found on the Yun-gui plateau (Southwest China), a low-latitude (∼23.3–26.6° N) and high-altitude (∼2000 m) region with acidic soil (pH 4.5∼5.5)), genes (107) related to "metal ion (including potassium, iron, and zinc) binding and transport, cell membrane function, and response to toxins" were found to be overrepresented.  These scientists found that expression of a phenotype encoded by these genes would be "consistent with functions in overcoming cation deficiency and aluminum toxicity that are common in acidic soils."  For the YT group, characterized by high temperature and high humidity areas in middle and lower regions of the Yangtze river, the researchers found "high enrichments of the LRR domain (24) genes," as well as other genes related to stress responses (40 genes), in comparison to other groups.

The paper also reported on genes correlated with altitude and adaptations to altitude.

These include 2755 association SNPs, involving 2408 genes, of which 1670 association SNPs were unique, and ∼51.9% of the associations were shared across different types of environmental variable (EVs), which suggested to them that different EVs may shape the same genomic regions.  These "hotspots" were located at "the top and bottom of Chromosome 2," wherein the top was enriched with genes encoding NBS-LRR proteins and the bottom was highly enriched with genes associated with responses to a series of stresses, as well as at the top of Chromosome 4, which was "highly enriched with genes associated encoding LRR domain-containing proteins."  Overall, a "total of 75 genomic loci associated with more than five EVs were identified and these loci were highly enriched with genes in stress-related pathways, such as plant–pathogen interaction, MAPK signaling pathway, response to stress, and defense response."

Related to specific stresses, the researchers found that "genes involved in ion transport were highly enriched in those associated with soil pH . . ., as soil pH affects absorption of metal ions in plants."  Also enriched were genes associated with "programmed cell death (PCD), innate immune response, plant–pathogen interaction, and DNA repair," as well as "secondary metabolism, including flavonoid metabolic process, jasmonic acid (JA) biosynthesis, and plant hormone signal transduction."  These researchers also identified "temperature-associated SNPs [including] 10 association hotspots on Chromosomes 1, 2, 3, 4, 5, 6, and 7 for more than eight EVs related to temperature and altitude."  These findings were significant, according to the authors, because "[t]olerance to low temperature in winter is a major factor that restricts the spread of peach to extremely cold regions."  One particular SNP identified in this way was "located in the gene PpAHP5" which is part of a gene cluster encoding six histidine phosphotransfer proteins (AHP); these have been "reported to be involved in mediating cold signaling" and was found to be "up-regulated by cold," and cold-resistant cultivars expressed the gene at higher levels than cold-sensitive ones.  Similarly, the researchers reported precipitation-related "hotspots"; one of these showed enrichment of several stress response related genes.

Of economic importance, the level of fruit produced by cultivars adapted to different environmental conditions was determined, and the researchers reported finding differential expression of genes resulting in higher soluble sugar content.  Environmental factors associated with this phenotype include aridity, high diurnal temperature variation, and long sunshine duration, according to these scientists, leading them to conclude that "higher soluble sugar content in accessions from northwestern China represents an adaptive trait driven by the local drought environment."

Other phenotypes include fruit flesh color, which the researchers found "strong geographic patterns, with ∼80% of yellow-fleshed peach landraces originating from northwestern China" and to be related to carotenoid metabolism (particularly overexpression of genes associated with carotenoid synthesis and loss-of-function mutations leading to carotenoid accumulation).  Studies of genetic variants associated with high altitude (Tibet) adapted cultivars were also reported, including specific patterns of gene expression, enrichment and loss (339 genomic regions, harboring 743 genes for high altitude cultivars, including ones involved in flavonoid production and UVB protection, and morphological changes in stomata thought to be involved in resistance to high-altitude hypoxia).

The depth of genetic information and environmental factor correlations (much more extensive than set forth in this synopsis) led these researcher to conclude:

In summary, this study provides new insights into peach adaptation to its habitat and how climate has shaped the genome of a perennial tree through natural selection.  These results also provide a new resource for studies of peach evolutionary biology and breeding, especially with regard to enhancing stress resistance.

* Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences; National Horticulture Germplasm Resources Center, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences; Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture & Forestry Sciences, Huazhong Agricultural University;  Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences;  IRTA–Centre de Recerca en Agrigenòmica (CSIC-IRTA-UAB-UB); Boyce Thompson Institute for Plant Research, Cornell University; U.S. Department of Agriculture– Agricultural Research Service, Robert W. Holley Center for Agriculture and Health

Giraffe Genome Reveals Relevant Adaptations

By Kevin E. Noonan --

The giraffe is the tallest extant terrestrial animal, and its iconic long neck (6 feet) provides advantages for foraging for food and detecting predators on the veldt over long distances.  As a consequence, however, the giraffe has a blood pressure two-fold higher than other ruminant animals needed to bring blood to the brain that is so far away from the animal's heart.

An earlier (2016) study of giraffe genome and genome of the related okapi (Okapia johnstoni) was not optimal, being restricted to 17,210 genes identified by comparison to cattle (Bos taurus) genome.  Last Wednesday, a team of Chinese and Norse scientists published a paper entitled "A towering genome: Experimentally validated adaptations to high blood pressure and extreme stature in the giraffe," in Science Advances.  In this paper, the authors described their work on Rothschild's giraffe (Giraffa camelopardalis rothschildi), which provided a higher "completeness" for this genome than previous studies.  The analysis provided the sequence of a 2.44 Gb assembly covering about 98% of giraffe genomic DNA.  Using comparisons with genomic DNA of cattle, goat, and okapi (with sperm whale as "outgroup"), the authors reported an assemblage of a putative common ancestor between giraffe and cattle; this resulted in a prediction of an evolutionary history of 4 chromosome fissions and 17 fusions that has resulted in the 15 haploid chromosome complement of modern giraffes (albeit admitting the need for further analysis to understand the significance of this result).

In this chromosomal complement these researchers discerned 101 genes under positive selection and 359 undergoing "rapid evolution," related to growth and development, nervous and visual systems, circadian rhythm, and blood pressure regulation.  One gene in particular was the focus this research:  the giraffe fibroblast growth factor receptor-like protein 1 (FGFRL1) gene was found to have seven non-synonymous mutations affecting the FGF binding domain, the most in any genetic sequence comparison to other ruminant mammals.  This gene was known to be involved in bone mineral density and hypertension resistance (thought to permit blood flow to the heights the giraffe's neck requires).  To investigate the relevance of these mutations to giraffe physiology, the researchers used CRISPR-Cas9 to introduce these mutations into the mouse FGFRL1 gene.  The resulting phenotype in these mice was found to be resistant to treatment with a high blood pressure-inducing drug (angiotensin II) while showing no developmental changes in cardiac structure; wildtype mice showed "significantly increased blood pressure" as a result of drug treatment.  These genetically altered mice also displayed significantly higher bone density as adults, with skeletal hypoplasia immediately postnatally.  Otherwise no deleterious effects were noted in these mice.  Together these results suggested these mutations could be responsible, at least in part, for the giraffe's characteristic long-necked phenotype.

This genetic research also detected differences in genes involving cardiac development, blood vessel characteristics and increased glomerular filtration rate in giraffe kidney, as well as genes involved in platelet function (including the phosphatidylinositol metabolism genes PIP4K2A, ISYNA1, MTMR3, CDS1, and INPP1) and ion transport related to cardiac contraction.

With regard to genetic adaptation of sensory genes (important for an herbivorous ungulate subject to predation by, inter alia, lions), these researchers found differences in genes related to eye development, vision, hearing, and balance.  Like other ruminants, giraffes have only two opsin genes, suggesting the absence of trichromatic color vision.  Perhaps curiously, the giraffe genome has lost 53 olfactory-related genes compared with the opaki, due to segmental deletion of sets of genes spatially clustered in the opaki genome, which these researchers speculated was a sensory "tradeoff" with their increased visual acuity.

Giraffes are known to have sleep durations among the lowest of all mammals.  Consistent with this phenotype circadian rhythm genes are altered in giraffes, particularly having a translation stop codon in the PER1 gene involved in circadian rhythm maintenance.

These researchers conclude their paper saying:

Overall, these results show that pleiotropy is a plausible mechanism for contributing to the suite of co-adaptations necessary in the evolution of the giraffe's towering stature.  However, because of the complexity of cardiovascular and sensory systems, more research on the functional consequences of giraffe-specific genetic variants is needed.

* School of Ecology and Environment, Northwestern Polytechnical University, Xi'an, China; Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi'an, China; College of Animal Science and Technology, Jilin Agricultural University, Changchun, China. BGI-Qingdao, BGI-Shenzhen, Qingdao, China; Shaanxi Key Laboratory for Animal Conservation, College of Life Sciences, Xi'an, China; Jiaxing SynBioLab. Co. Ltd., Jiaxing, China;  Research Center of Traditional Chinese Medicine, The Affiliated Hospital to Changchun University of Chinese Medicine, Changchun, China; Center for Evolutionary Hologenomics, GLOBE Institute, University of Copenhagen, Øster Voldgade, Copenhagen, Denmark; Norwegian University of Science and Technology, University Museum, 7491 Trondheim, Norway; Section for Computational and RNA Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China.

Image of Rothschild's giraffe (Giraffa camelopardalis rothschildi) at Murchison Falls NP, UGANDA by Bernard DUPONT, from the Wikimedia Commons under the Creative Commons Attribution-ShareAlike 2.0 Generic license.