•This brief review was presented here to facilitate an independent and more balanced discussion on the potential risks due to the presence of adenovirus vector DNA (AstraZeneca, Johnson & Johnson, ...Sputnik V and others) or SARS-CoV-2 RNA (BioNTech/Pfizer, Moderna) in vaccines that are supposed to protect against Covid-19. Of course, injections of vector-based vaccines into human deltoid muscle is a different matter than rare chance events leading to recombination events between foreign and human DNAs in experimental systems as described above. Moreover, neither type nor frequency of consequences of rare vector integration events can be realistically assessed at present. The recently published results on the benefits of protection against Covid-19 offered by the BioNTech/Pfizer vaccines are encouraging Dagan et al. 2021. Granted, the jury is still out on whether any of the vaccines’ will protect against the more dangerous new SARS-CoV-2 variants from the UK, South Africa, Brazil and unknown variants that might arise in the future given the poorly controlled levels of viral replication around the world. Lastly, we are ignorant about vaccine protection against the development of prolonged and late-onset symptoms of Covid-19.•The information presented in this review will help future vaccinees to weigh a risk versus benefit assessment, namely the integration events of adenovirus vector or of SARS-CoV-2 RNA reverse transcript DNA at low frequency versus hopefully high vaccine efficacy and protection. Moreover, since SARS-CoV-2 infection by itself can be associated with the integration of reverse transcripts of the viral RNA Zhang et al, 2020, this series of events might become inescapable in any SARS-CoV-2 infection. Lastly, the extent to which adenoviral gene products might become co-expressed with the SARS-CoV-2 spike glycoprotein upon vector-vaccine injection into human deltoid muscles remains un-investigated. At present we cannot gauge their possible effects on the human organism, if actually expressed. Opportunities and risks, both at the same time, remain beyond our expectations of absolute controls because life and evolution likely have been based on “chance mechanisms” from the very beginning. Clinical observations on long lasting positive RT-PCR test results that imply SARS-CoV-2 DNA integration into the human genome in the course of some Covid-19 cases, render apprehensions about vaccine-associated integration events unrealistic, when compared to the hoped-for benefits by vaccination against Covid-19. The human population of 2021 faces a biomedical crisis of unprecedented dimensions in recent times and will have to accept the best available countermeasures against Covid-19 of the day – Vaccination.
Vigorous vaccination programs against SARS-CoV-2-causing Covid-19 are the major chance to fight this dreadful pandemic. The currently administered vaccines depend on adenovirus DNA vectors or on SARS-CoV-2 mRNA that might become reverse transcribed into DNA, however infrequently. In some societies, people have become sensitized against the potential short- or long-term side effects of foreign DNA being injected into humans. In my laboratory, the fate of foreign DNA in mammalian (human) cells and organisms has been investigated for many years. In this review, a summary of the results obtained will be presented. This synopsis has been put in the evolutionary context of retrotransposon insertions into pre-human genomes millions of years ago. In addition, studies on adenovirus vector-based DNA, on the fate of food-ingested DNA as well as the long-term persistence of SARS-CoV-2 RNA/DNA will be described. Actual integration of viral DNA molecules and of adenovirus vector DNA will likely be chance events whose frequency and epigenetic consequences cannot with certainty be assessed. The review also addresses problems of remaining adenoviral gene expression in adenoviral-based vectors and their role in side effects of vaccines. Eventually, it will come down to weighing the possible risks of genomic insertions of vaccine-associated foreign DNA and unknown levels of vector-carried adenoviral gene expression versus protection against the dangers of Covid-19. A decision in favor of vaccination against life-threatening disease appears prudent. Informing the public about the complexities of biology will be a reliable guide when having to reach personal decisions about vaccinations.
Scientists and the public were alarmed at the first large viral variant of SARS‐CoV‐2 reported in December 2020. We have followed the time course of emerging viral mutants and variants during the ...SARS‐CoV‐2 pandemic in ten countries on four continents. We examined > 383,500 complete SARS‐CoV‐2 nucleotide sequences in GISAID (Global Initiative of Sharing All Influenza Data) with sampling dates extending until April 05, 2021. These sequences originated from ten different countries: United Kingdom, South Africa, Brazil, United States, India, Russia, France, Spain, Germany, and China. Among the 77 to 100 novel mutations, some previously reported mutations waned and some of them increased in prevalence over time. VUI2012/01 (B.1.1.7) and 501Y.V2 (B.1.351), the so‐called UK and South Africa variants, respectively, and two variants from Brazil, 484K.V2, now called P.1 and P.2, increased in prevalence. Despite lockdowns, worldwide active replication in genetically and socio‐economically diverse populations facilitated selection of new mutations. The data on mutant and variant SARS‐CoV‐2 strains provided here comprise a global resource for easy access to the myriad mutations and variants detected to date globally. Rapidly evolving new variant and mutant strains might give rise to escape variants, capable of limiting the efficacy of vaccines, therapies, and diagnostic tests.
Synopsis
This 2020/21 time course study shows the rapid rise of new SARS‐CoV‐2 mutants and variants across the entire genome during worldwide viral replication. In 10 countries, 40 to 65% of mutants were C to T transitions. Viral mutations will affect vaccination programs.
We analyzed > 383,500 SARS‐CoV‐2 RNA sequences for the occurrence of mutations across the entire genome. The time course of mutations emerging between 01/2020 and 03/2021 was determined.
We initially identified ~ 10 prevalent mutations. About 77 to 100 new mutations arose concomitant with the spread of Covid‐19 between March/April 2020 and January 2021, followed by the emergence of variants in December 2020.
A study of the pathogenicity of viral mutations will help understand Covid‐19 outbreaks and symptoms. Monitoring mutant selection will aid Covid‐19 diagnosis, vaccine development and therapy. New mutants will compromise vaccine efficiency.
Among the SARS‐CoV‐2 mutants, C to U transitions at a frequency between 40 to 65% were prevalent. Cellular cytosine deaminases, possibly of the APOBEC type, likely drive viral mutagenesis.
This 2020/21 time course study shows the rapid rise of new SARS‐CoV‐2 mutants and variants across the entire genome during worldwide viral replication. In 10 countries, 40 to 65% of mutants were C to T transitions. Viral mutations will affect vaccination programs.
A synopsis will be presented of work on DNA methylation, the first epigenetic signal to be recognized. In the author´s laboratory, the following problems dealing with DNA methylation have been ...addressed over the past 32 years:(1) The de novo methylation of foreign DNA integrated into mammalian genomes. (2) Inverse correlations between promoter methylation and activity.(3) The long-term inactivating effect of site-specific promoter methylation. (4) Adenovirus E1 functions in trans and a strong enhancer in cis cancel the silencing effect of promoter methylation.(5) Frog virus 3, an iridovirus with a completely CpG-methylated genome. (6) Mechanisms of de novo methylation.(7) Different segments of the genome possess topical methylation memories.(8) Consequences of foreign DNA insertion into mammalian genomes: alterations of DNA methylation in cis and trans.(9) The epigenetic status of an adenovirus transgenome in Ad12-transformed hamster cells. (10) Cell type-specific patterns of DNA methylation: interindividual concordance in the human genome.
Apart from its well-documented role in long-term promoter silencing, the genome-wide distribution patterns of ~ 28 million methylated or unmethylated CpG dinucleotides, e. g. in the human genome, is ...in search of genetic functions. We have set out to study changes in the cellular CpG methylation profile upon introducing foreign DNA into mammalian cells. As stress factors served the genomic integration of foreign (viral or bacterial plasmid) DNA, virus infections or the immortalization of cells with Epstein Barr Virus (EBV). In all instances investigated, alterations in cellular CpG methylation and transcription profiles were observed to different degrees. In the case of adenovirus DNA integration in adenovirus type 12 (Ad12)-transformed hamster cells, the extensive changes in cellular CpG methylation persisted even after the complete loss of all transgenomic Ad12 DNA. Hence, stress-induced alterations in CpG methylation can be inherited independent of the continued presence of the transgenome. Upon virus infections, changes in cellular CpG methylation appear early after infection. In EBV immortalized as compared to control cells, CpG hypermethylation in the far-upstream region of the human FMR1 promoter decreased four-fold. We conclude that in the wake of cellular stress due to foreign DNA entry, preexisting CpG methylation patterns were altered, possibly at specific CpG dinucleotides. Frequently, transcription patterns were also affected. As a working concept, we view CpG methylation profiles in mammalian genomes as a guarding sensor for genomic stability under epigenetic control. As a caveat towards manipulations of cells with foreign DNA, such cells can no longer be considered identical to their un-manipulated counterparts.
The emerging Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its variants have raised tantalizing questions about evolutionary mechanisms that continue to shape biology today. We ...have compared the nucleotide sequence of SARS-CoV-2 RNA to that of genomes of many different viruses, of endosymbiotic proteobacterial and bacterial DNAs, and of human mitochondrial DNA. The entire 4,641,652 nt DNA sequence of Escherichia coli K12 has been computer-matched to SARS-CoV-2 RNA. Numerous, very similar micro-modular clusters of 3 to 13 nucleotides lengths were detected with sequence identities of 40 to >50% in specific genome segments between SARS-CoV-2 and the investigated genomes. These clusters were part of patch-type homologies. Control sequence comparisons between 1000 randomly computer-composed sequences of 29.9 kb and with the A, C, G, T base composition of SARS-CoV-2 genome versus the reference Wuhan SARS-CoV-2 sequence showed similar patterns of sequence homologies. The universal A, C, G, T genetic coding mode might have succeeded in evolution due in part to its built-in capacity to select for a substantial reservoir of micro-modular domains and employ them as platforms for integrative recombination. Their role in SARS-CoV-2 interspecies transition and the generation of variants appears likely, but their actual involvement will require detailed investigations.
We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by ...bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.
The human genome segment upstream of the FMR1 (fragile X mental retardation 1) gene (Xq27.3) contains several genetic signals, among them is a DNA methylation boundary that is located 65–70 CpGs ...upstream of the CGG repeat. In fragile X syndrome (FXS), the boundary is lost, and the promoter is inactivated by methylation spreading. Here we document boundary stability in spite of critical expansions of the CGG trinucleotide repeat in male or female premutation carriers and in high functioning males (HFMs). HFMs carry a full CGG repeat expansion but exhibit an unmethylated promoter and lack the FXS phenotype. The boundary is also stable in Turner (45, X) females. A CTCF-binding site is located slightly upstream of the methylation boundary and carries a unique G-to-A polymorphism (single nucleotide polymorphism), which occurs 3.6 times more frequently in genomes with CGG expansions. The increased frequency of this single nucleotide polymorphism might have functional significance. In CGG expansions, the CTCF region does not harbor additional mutations. In FXS individuals and often in cells transgenomic for EBV (Epstein Barr Virus) DNA or for the telomerase gene, the large number of normally methylated CpGs in the far-upstream region of the boundary is decreased about 4-fold. A methylation boundary is also present in the human genome segment upstream of the HTT (huntingtin) promoter (4p16.3) and is stable both in normal and Huntington disease chromosomes. Hence, the vicinity of an expanded repeat does not per se compromise methylation boundaries. Methylation boundaries exert an important function as promoter safeguards.
The focus of the current project has been on the structure, function, and stability of DNA methylation boundaries in the vicinity of stable and/or unstable trinucleotide repeats. We have now documented that, in at-risk CGG expansions, including those in the genomes of premutation carriers with 50–200 repeats or of HFMs with full expansions beyond the 200-repeat threshold and up to 400 repeats, the methylation boundary is preserved and the FMR1 promoter (Xq27.3) is protected against de novo methylation. Our methylation analyses of human promoters located in the vicinity of trinucleotide repeats have been extended to the promoter of the HTT gene (4p16.3). About 800 nucleotides 5′-upstream of the HTT gene promoter, there is a characteristic, equally distinct methylation boundary as in the FMR1 promoter. The HTT boundary remains unaltered in Huntington disease genomes with CAG expansions of n>40. Our data suggest that trinucleotide repeat expansions in the vicinity of methylation boundaries do not lead per se to the breakdown of these boundaries as in FXS patients. Display omitted
•How is the stability of promoters in the human genome protected?•DNA methylation boundaries upstream of the human FMR1 and HTT promoter sequences.•Boundary stability even in the presence of trinucleotide repeat expansions.•Loss of promoter far-upstream methylation profiles upon insertion of foreign DNA.•DNA methylation boundaries are important in safeguarding promoter stability.
In this article, a new concept for general pathogenesis has been proposed. Advances in molecular genetics have led to the realization that essential concepts in the framework of molecular biology are ...still missing. Clinical medicine is plagued by similar shortcomings: The questioning of current paradigms could open new vistas and invite challenging approaches. This article presents an unconventional idea. Foreign DNA which is regularly ingested with the essential food supply is not completely degraded. Small quantities of fragmented DNA rather persist transiently in the gastro-intestinal tract of mice and can be traced to various organ systems, except for cells in the germ line. Foreign DNA entering and persisting in mammalian cells can stochastically lead to genome-wide alterations of transcriptional and CpG DNA methylation profiles. In the course of food-ingested DNA invading somatic cells, completely new cell types can be generated which might be involved in the causation of common ailments. Projects emanating from this perception merit critical analysis and rigorous pursuit.
Since many research projects in biology, medicine and agriculture involve genome manipulations in mammalian or plant cells and organisms by transfecting foreign DNA with a variety of techniques and a ...gamut of highly efficient vector systems, the sequelae of these procedures need to be diligently investigated. There are reports about the success of these techniques, but caveats remain due to unexpected and possibly unforeseeable consequences. Since the long-term sequelae of the invasion of foreign DNA into cells or organisms have not been extensively studied, projections about the possible problems arising have remained guesswork for far too long. In some of these instances, the accidental activation of cellular oncogenes by inadvertently placing an inserted gene in the vicinity of the oncogenes’ promotors have been invoked as plausible but unproven explanations. ...it is likely that there may be more fundamental problems, like changes of the epigenome, in the wake of foreign DNA insertions that need further consideration and investigation. Critical investigations International groups of researchers could independently coordinate research along the following lines (Box 1): This strategy could be a first step toward a more rational discussion on the potential problems inherent in human gene therapy or GMOs in medicine and replace pure guesswork that has dominated this emotionalized debate for decades without reliable conclusions.
•Study of SARS-CoV-2 genomes from world-wide isolates.•Sequence comparisons of 570 isolates to original Wuhan 2019 SARS-CoV-2 clade.•Identification of several hotspot mutants after world-wide ...spreading of virus.•Several hotspot mutations affect sequences of replication-relevant viral proteins.•How do hotspot mutations relate to viral pathogenicity?
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first identified in Wuhan, China late in 2019. Nine months later (Sept. 23, 2020), the virus has infected > 31.6 million people around the world and caused > 971.000 (3.07 %) fatalities in 220 countries and territories. Research on the genetics of the SARS-CoV-2 genome, its mutants and their penetrance can aid future defense strategies. By analyzing sequence data deposited between December 2019 and end of May 2020, we have compared nucleotide sequences of 570 SARS-CoV-2 genomes from China, Europe, the US, and India to the sequence of the Wuhan isolate. During worldwide spreading among human populations, at least 10 distinct hotspot mutations had been selected and found in up to > 80 % of viral genomes. Many of these mutations led to amino acid exchanges in replication-relevant viral proteins. Mutations in the SARS-CoV-2 genome would also impinge upon the secondary structure of the viral RNA molecule and its repertoire of interactions with essential cellular and viral proteins. The increasing frequency of SARS-CoV-2 mutation hotspots might select for dangerous viral pathogens. Alternatively, in a 29.900 nucleotide-genome, there might be a limit to the number of mutable and selectable sites which, when exhausted, could prove disadvantageous to viral survival. The speed, at which novel SARS-CoV-2 mutants are selected and dispersed around the world, could pose problems for the development of vaccines and therapeutics.
PDF
