Somatic mutation patterns at Ig and Non-Ig Loci.

The reverse transcriptase (RT) model of immunoglobulin (Ig) somatic hypermutation (SHM) has received insufficient scientific attention. This is understandable given that DNA deamination mediated by activation-induced deaminase (AID), the initiating step of Ig SHM, has dominated experiments since 2002. We summarise some key history of the RT Ig SHM model dating to 1987. For example, it is now established that DNA polymerase η, the sole DNA repair polymerase involved in post-replication short-patch repair, is an efficient cellular RT. This implies that it is potentially able to initiate target site reverse transcription by RNA-directed DNA repair at AID-induced lesions. Recently, DNA polymerase θ has also been shown to be an efficient cellular RT. Since DNA polymerase θ plays no significant role in Ig SHM, it could serve a similar RNA-dependent DNA polymerase role as DNA polymerase η at non-Ig loci in the putative RNA-templated nucleotide excision repair of bulky adducts and other mutagenic lesions on the transcribed strand. A major yet still poorly recognised consequence of the proposed RT process in Ig SHM is the generation of significant and characteristic strand-biased mutation signatures at both deoxyadenosine/deoxythymidine and deoxyguanosine/deoxycytidine base pairs. In this historical perspective, we highlight how diagnostic strand-biased mutation signatures are detected in vivo during SHM at both Ig loci in germinal centre B lymphocytes and non-Ig loci in cancer genomes. These strand-biased signatures have been significantly obscured by technical issues created by improper use of the polymerase chain reaction technique. A heightened awareness of this fact should contribute to better data interpretation and somatic mutation pattern recognition both at Ig and non-Ig loci.

Analysis of SARS-CoV-2 haplotypes and genomic sequences during 2020 in Victoria, Australia, in the context of putative deficits in innate immune deaminase anti-viral responses.

The SARS-CoV-2 epidemic infections in Australia during 2020 were small in number in epidemiological terms and are well described. The SARS-CoV-2 genomic sequence data of many infected patients have been largely curated in a number of publicly available databases, including the corresponding epidemiological data made available by the Victorian Department of Health and Human Services. We have critically analysed the available SARS-CoV-2 haplotypes and genomic sequences in the context of putative deficits in innate immune APOBEC and ADAR deaminase anti-viral responses. It is now known that immune impaired elderly co-morbid patients display clear deficits in interferon type 1 (α/ß) and III (λ) stimulated innate immune gene cascades, of which APOBEC and ADAR induced expression are part. These deficiencies may help explain some of the clear genetic patterns in SARS-CoV-2 genomes isolated in Victoria, Australia, during the 2nd Wave (June-September, 2020). We tested the hypothesis that predicted lowered innate immune APOBEC and ADAR anti-viral deaminase responses in a significant proportion of elderly patients would be consistent with/reflected in a low level of observed mutagenesis in many isolated SARS-CoV-2 genomes. Our findings are consistent with this expectation. The analysis also supports the conclusions of the Victorian government's Department of Health that essentially one variant or haplotype infected Victorian aged care facilities where the great majority (79%) of all 820 SARS-CoV-2 associated deaths occurred. The implications of our data analysis for other localized epidemics and efficient coronavirus vaccine design and delivery are discussed.

Nature of Acquired Immune Responses, Epitope Specificity and Resultant Protection from SARS-CoV-2.

The primary global response to the SARS-CoV-2 pandemic has been to bring to the clinic as rapidly as possible a number of vaccines that are predicted to enhance immunity to this viral infection. While the rapidity with which these vaccines have been developed and tested (at least for short-term efficacy and safety) is commendable, it should be acknowledged that this has occurred despite the lack of research into, and understanding of, the immune elements important for natural host protection against the virus, making this endeavor a somewhat unique one in medical history. In contrast, as pointed out in the review below, there were already important past observations that suggested that respiratory infections at mucosal surfaces were susceptible to immune clearance by mechanisms not typical of infections caused by systemic (blood-borne) pathogens. Accordingly, it was likely to be important to understand the role for both innate and acquired immunity in response to viral infection, as well as the optimum acquired immune resistance mechanisms for viral clearance (B cell or antibody-mediated, versus T cell mediated). This information was needed both to guide vaccine development and to monitor its success. We have known that many pathogens enter into a quasi-symbiotic relationship with the host, with each undergoing sequential change in response to alterations the other makes to its presence. The subsequent evolution of viral variants which has caused such widespread concern over the last 3-6 months as host immunity develops was an entirely predictable response. What is still not known is whether there will be other unexpected side-effects of the deployment of novel vaccines in humans which have yet to be characterized, and, if so, how and if these can be avoided. We conclude by remarking that to ignore a substantial body of well-attested immunological research in favour of expediency is a poor way to proceed.

Predicting clinical outcomes using cancer progression associated signatures.

Somatic mutation signatures are an informative facet of cancer aetiology, however they are rarely useful for predicting patient outcome. The aim of this study is to evaluate the utility of a panel of 142 mutation-signature-associated metrics (P142) for predicting cancer progression in patients from a 'TCGA PanCancer Atlas' cohort. The P142 metrics are comprised of AID/APOBEC and ADAR deaminase associated SNVs analyzed for codon context, strand bias, and transitions/transversions. TCGA tumor-normal mutation data was obtained for 10,437 patients, representing 31 of the most prevalent forms of cancer. Stratified random sampling was used to split patients into training, tuning and validation cohorts for each cancer type. Cancer specific machine learning (XGBoost) models were built using the output from the P142 panel to predict patient Progression Free Survival (PFS) status as either "High PFS" or "Low PFS". Predictive performance of each model was evaluated using the validation cohort. Models accurately predicted PFS status for several cancer types, including adrenocortical carcinoma, glioma, mesothelioma, and sarcoma. In conclusion, the P142 panel of metrics successfully predicted cancer progression status in patients with some, but not all cancer types analyzed. These results pave the way for future studies on cancer progression associated signatures.

The efficient Lamarckian spread of life in the cosmos.

In this Chapter we discuss the various mechanisms that are available for the possible transfer of cosmic microbial living systems from one cosmic habitat to another. With the 100 or so habitable planets that are now known to exist in our galaxy alone transfers of cometary dust carrying life including fragments of icy planetoids/asteroids would be expected to occur on a routine basis. It is thus easy to view the galaxy as a single connected "biosphere" of which our planet Earth is a minor component. The Hoyle-Wickramasinghe Panspermia paradigm provides a cogent biological rationale for the actual widespread existence of Lamarckian modes of inheritance in terrestrial systems (which we review here). Thus the Panspermia paradigm provides the raison d'etre for Lamarckian Inheritance. Under a terrestrially confined neoDarwinian viewpoint such an association may have been thought spurious in the past. Our aim here is to outline the main evidence for rapid terrestrial-based Lamarckian-based evolutionary hypermutation processes dependent on reverse transcription-coupled mechanisms among others. Such rapid adaptation mechanisms would be consistent with the effective cosmic spread of living systems. For example, a viable, or cryo-preserved, living system traveling through space in a protective matrix will of necessity need to adapt rapidly and proliferate on landing in a new cosmic niche. Lamarckian mechanisms thus come to the fore and supersede the slow (blind and random) genetic processes expected under neoDarwinian Earth centred theories.

The mutagenic source and power of our own evolution.

This chapter addresses the molecular mechanism and source of the numerous mutagenic changes that genomes, particularly mammalian genomes, can undergo during normal development and in diseased states. The central role of pathogen and other disease-inducing innate immunity via the action of the cytosine (AID/APOBEC) and adenosine (ADAR) deaminases is reviewed in some depth. The general and universal nature of deaminase-mediated mutagenesis is an important key to understanding cosmic genetic evolutionary processes.

Origin of new emergent Coronavirus and Candida fungal diseases-Terrestrial or cosmic?

The origins and global spread of two recent, yet quite different, pandemic diseases is discussed and reviewed in depth: Candida auris, a eukaryotic fungal disease, and COVID-19 (SARS-CoV-2), a positive strand RNA viral respiratory disease. Both these diseases display highly distinctive patterns of sudden emergence and global spread, which are not easy to understand by conventional epidemiological analysis based on simple infection-driven human- to-human spread of an infectious disease (assumed to jump suddenly and thus genetically, from an animal reservoir). Both these enigmatic diseases make sense however under a Panspermia in-fall model and the evidence consistent with such a model is critically reviewed.

Blood and saliva-derived exomes from healthy Caucasian subjects do not display overt evidence of somatic mosaicism.

Somatic mosaicism is a normal occurrence during development in the tissues and organs. As part of establishing a "healthy population "(HP) background or base-line, we investigated whether such mosaicism can be routinely detected in the circulating DNA secured from a rigorously designed healthy human liquid biopsy clinical trial (saliva, blood). We deployed next generation (NG) whole exome sequencing (WES) at median exome coverage rates of 97.2 % (-to-30x) and 70.0 % (-to-100x). We found that somatic mosaicism is not detectable by such standard bulk WES sequencing assays in saliva and blood DNA in 24 normal healthy Caucasians of both sexes from 18 to 60 years of age. We conclude that for circulating DNA using standard WES no novel somatic mutational variants can be detected in protein-coding regions of normal healthy subjects. This implies that the extent within normal tissues of somatic mosaicism must be at a lower level, below the detection threshold, for these circulating DNA WES read depths.

A proposed reverse transcription mechanism for (CAG)n and similar expandable repeats that cause neurological and other diseases.

The mechanism of (CAG)n repeat generation, and related expandable repeat diseases in non-dividing cells, is currently understood in terms of a DNA template-based DNA repair synthesis process involving hairpin stabilized slippage, local error-prone repair via MutSß (MSH2-MSH3) hairpin protective stabilization, then nascent strand extension by DNA polymerases-ß and -δ. We advance a very similar slipped hairpin-stabilized model involving MSH2-MSH3 with two key differences: the copying template may also be the nascent pre-mRNA with the repair pathway being mediated by the Y-family error-prone enzymes DNA polymerase-η and DNA polymerase-κ acting as reverse transcriptases. We argue that both DNA-based and RNA-based mechanisms could well be activated in affected non-dividing brain cells in vivo. Here, we compare the advantages of the RNA/RT-based model proposed by us as an adjunct to previously proposed models. In brief, our model depends upon dysregulated innate and adaptive immunity cascades involving AID/APOBEC and ADAR deaminases that are known to be involved in normal locus-specific immunoglobulin somatic hypermutation, cancer progression and somatic mutations at many off-target non-immunoglobulin sites across the genome: we explain how these processes could also play an active role in repeat expansion diseases at RNA polymerase II-transcribed genes.

Regulatory T cells and co-evolution of allele-specific MHC recognition by the TCR.

What is the evolutionary mechanism for the TCR-MHC-conserved interaction? We extend Dembic's model (Dembic Z. In, Scand J Immunol e12806, 2019) of thymus positive selection for high-avidity anti-self-MHC Tregs among double (CD4 + CD8+)-positive (DP) developing thymocytes. This model is based on competition for self-MHC (+ Pep) complexes presented on cortical epithelium. Such T cells exit as CD4 + CD25+FoxP3 + thymic-derived Tregs (tTregs). The other positively selected DP T cells are then negatively selected on medulla epithelium removing high-avidity anti-self-MHC + Pep as T cells commit to CD4 + or CD8 + lineages. The process is likened to the competitive selection and affinity maturation in Germinal Centre for the somatic hypermutation (SHM) of rearranged immunoglobulin (Ig) variable region (V[D]Js) of centrocytes bearing antigen-specific B cell receptors (BCR). We now argue that the same direct SHM processes for TCRs occur in post-antigenic Germinal Centres, but now occurring in peripheral pTregs. This model provides a potential solution to a long-standing problem previously recognized by Cohn and others (Cohn M, Anderson CC, Dembic Z. In, Scand J Immunol e12790, 2019) of how co-evolution occurs of species-specific MHC alleles with the repertoire of their germline TCR V counterparts. We suggest this is not by 'blind', slow, and random Darwinian natural selection events, but a rapid structured somatic selection vertical transmission process. The pTregs bearing somatic TCR V mutant genes then, on arrival in reproductive tissues, can donate their TCR V sequences via soma-to-germline feedback as discussed in this journal earlier. (Steele EJ, Lindley RA. In, Scand J Immunol e12670, 2018) The high-avidity tTregs also participate in the same process to maintain a biased, high-avidity anti-self-MHC germline V repertoire.

Lamarck and Panspermia - On the Efficient Spread of Living Systems Throughout the Cosmos.

We review the main lines of evidence (molecular, cellular and whole organism) published since the 1970s demonstrating Lamarckian Inheritance in animals, plants and microorganisms viz. the transgenerational inheritance of environmentally-induced acquired characteristics. The studies in animals demonstrate the genetic permeability of the soma-germline Weismann Barrier. The widespread nature of environmentally-directed inheritance phenomena reviewed here contradicts a key pillar of neo-Darwinism which affirms the rigidity of the Weismann Barrier. These developments suggest that neo-Darwinian evolutionary theory is in need of significant revision. We argue that Lamarckian inheritance strategies involving environmentally-induced rapid directional genetic adaptations make biological sense in the context of cosmic Panspermia allowing the efficient spread of living systems and genetic innovation throughout the Universe. The Hoyle-Wickramasinghe Panspermia paradigm also developed since the 1970s, unlike strictly geocentric neo-Darwinism provides a cogent biological rationale for the actual widespread existence of Lamarckian modes of inheritance - it provides its raison d'être. Under a terrestrially confined neo-Darwinian viewpoint such an association may have been thought spurious in the past. Our aim is to outline the conceptual links between rapid Lamarckian-based evolutionary hypermutation processes dependent on reverse transcription-coupled mechanisms among others and the effective cosmic spread of living systems. For example, a viable, or cryo-preserved, living system travelling through space in a protective matrix will need of necessity to rapidly adapt and proliferate on landing in a new cosmic niche. Lamarckian mechanisms thus come to the fore and supersede the slow (blind and random) genetic processes expected under a traditional neo-Darwinian evolutionary paradigm.

Molecular model linking Th2 polarized M2 tumour-associated macrophages with deaminase-mediated cancer progression mutation signatures.

A new and diverse range of somatic mutation signatures are observed in late-stage cancers, but the underlying reasons are not fully understood. We advance a "combinatorial association model" for deaminase binding domain (DBD) diversification to explain the generation of previously observed cancer-progression associated mutation signatures. We also propose that changes in the polarization of tumour-associated macrophages (TAMs) are accompanied by the expression of deaminases with a new and diverse range of DBDs, and thus accounting for the generation of new somatic mutation signatures. The mechanism proposed is molecularly reminiscent of combinatorial association of heavy (H) and light (L) protein chains following V(D)J recombination of immunoglobulin molecules (and similarly for protein chains in heterodimers α/ß and γ/δ of V(D)Js of T Cell Receptors) required for pathogen antigen recognition by B cells and T cells, respectively. We also discuss whether extracellular vesicles (EVs) emanating from tumour enhancing M2-polarized macrophages represent a likely source of the de novo deaminase DBDs. We conclude that M2-polarized macrophages extruding EVs loaded with deaminase proteins or deaminase-specific transcription/translation regulatory factors and like information may directly trigger deaminase diversification within cancer cells, and thus account for the many new somatic mutation signatures that are indicative of cancer progression. This hypothesis now has a plausible evidentiary base, and it is worth direct testing in future investigations. A long-term objective would be to identify molecular biomarkers predicting cancer progression (or metastatic disease) and to support the development of new drug targets before metastatic pathways are activated.

APOBEC and ADAR deaminases may cause many single nucleotide polymorphisms curated in the OMIM database.

Cytosine and adenosine deamination events (DNA, RNA substrates) account for most codon-context Targeted Somatic Mutation (TSM) patterns observed in immunoglobulin (Ig) somatic hypermutation (SHM), and in cancer exomes following Ig-SHM-like responses. TSM refers to the process of somatic mutagenesis involving deamination events that results on a dominant type of mutation (e.g., C-to-T), and co-incident at a particular motif (e.g., WRC), and preferentially targeting the first, second or third nucleotide position within the mutated codon (e.g. MC1, MC2 or MC3, read 5-prime to 3-prime). It is now widely accepted that if left uncorrected, the accumulation of uncorrected TSMs involving the deaminases, may lead to a diagnosis of cancer or other degenerative disease. Our hypothesis is that many missense, nonsense and synonymous single nucleotide polymorphisms (SNPs) associated with clinically significant diseases may have arisen in the population by similar highly targeted deamination events. The OMIM database was searched for disease-associated SNPs on the X chromosome, and for all chromosomes. The nucleotide substitution patterns for disease-associated SNPs were analyzed by the TSM method to identify the likely deaminase source for C-to-U (C-to-T/G-to-A) and A-to-I (A-to-G/T-to-C) derived gene mutations preferentially targeting known sequence motifs associated with the deaminases: AID, APOBEC3G, APOBEC3B and ADAR 1/2. Of the 789 OMIM SNPs analysed. In both data sets, the mutation targeting preferences within the mutated codon reveal a statistically significant bias (p < 0.001). The results imply that a deamination of C-site and A-site targets are written into the human germline for the chromosome wide exomic SNPs analysed. This is consistent with previously observed mutation patterns arising in cancer genomes and hypermutated Ig genes during SHM. The results imply that similar types of deaminase-mediated molecular processes that occur in somatic hypermutation and cancer, may be contributing causative drivers of human exomic SNPs.

ADAR deaminase A-to-I editing of DNA and RNA moieties of RNA:DNA hybrids has implications for the mechanism of Ig somatic hypermutation.

The implications are discussed of recently published biochemical studies on ADAR-mediated A-to-I DNA and RNA deamination at RNA:DNA hybrids. The significance of these data are related to previous work on strand-biased and codon-context mutation signatures in B lymphocytes and cancer genomes. Those studies have established that there are two significant strand biases at A:T and G:C base pairs, A-site mutations exceed T-site mutations (A>>T) by 2.9 fold and G-site mutations exceed C-site mutations (G>>C) by 1.7 fold. Both these strand biases are inconsistent with alternative "DNA Deamination" mechanisms, yet are expected consequences of the RNA/RT-based "Reverse Transcriptase" mechanism of immunoglobulin (Ig) somatic hypermutation (SHM). The A-to-I DNA editing component at RNA:DNA hybrids that is likely to occur in Transcription Bubbles, while important, is of far lower A-to-I editing efficiency than in dsRNA substrates. The RNA moiety of RNA:DNA hybrids is also edited at similar lower frequencies relative to the editing rate at dsRNA substrates. Further, if the A-to-I DNA editing at RNA:DNA hybrids were the sole cause of A-to-I (read as A-to-G) mutation events for Ig SHM in vivo then the exact opposite strand biases at A:T base pairs (T>>A) of what is actually observed (A>>T) would be predicted. It is concluded that the strand-biased somatic mutation patterns at both A:T and G:C base pairs in vivo are best interpreted by the sequential steps of the RNA/RT-based mechanism. Further, the direct DNA A-to-I deamination at Transcription Bubbles is expected to contribute to the T-to-C component of the strand-biased Ig SHM spectrum.

Association between targeted somatic mutation (TSM) signatures and HGS-OvCa progression.

Evidence already exists that the activation-induced cytidine deaminase (AID/APOBEC) and the adenosine deaminase (ADAR) families of enzymes are implicated as powerful mutagens in oncogenic processes in many somatic tissues. Each deaminase is identified by the DNA or RNA nucleotide sequence ("motif") surrounding the nucleotide targeted for deamination. The primary objective of this study is to develop an in silico approach to identify nucleotide sequence changes of the target motifs of key deaminases during oncogenesis. If successful, a secondary objective is to investigate if such changes are associated with disease progression indicators that include disease stage and progression-free survival time. Using a discovery cohort of 194 high-grade serous ovarian adenocarcinoma (HGS-OvCa) exomes, the results confirm the ability of the novel in silico approach used to identify changes in the preferred target motifs for AID, APOBEC3G, APOBEC3B, and ADAR1 during oncogenesis. Using this approach, a set of new cancer-progression associated signatures (C-PASs) were identified. Furthermore, it was found that the C-PAS identified can be used to differentiate between the cohort of patients that remained progression-free for longer than 60 months, from those in which disease progressed within 60 months (sensitivity 95%, specificity 90%). The spectrum of outcomes observed here could provide a foundation for future clinical assessment of susceptibility variants in ovarian, and several other cancers as disease progresses. The ability of the in silico methodology used to identify changes in deaminase motifs during oncogenesis also suggests new links between immune system function and tumorigenesis.

The importance of codon context for understanding the Ig-like somatic hypermutation strand-biased patterns in TP53 mutations in breast cancer.

Evidence already exists that the activation-induced deaminase (AID)/APOBEC family constitutes a set of differentially expressed enzymes capable of deaminating cytosines (C to U) in single-stranded DNA (ssDNA) and that they are potentially powerful mutagens. The mutagenic processes involved are believed to be activated in many nonlymphoid tissue types-for example, initiating some cancers and/or leading to further somatic mutagenesis. To investigate the extent that codon context might be important in influencing the likely location of TP53 mutations in breast cancer, the codon-bias patterns resulting from the ssDNA target specificities of cytidine deaminases of the AID/APOBEC family were analyzed. The data indicate that codon context strongly influences the likely location of mutations at motifs for AID/APOBEC1/APOBEC3G, and at WA sites. An unexpected finding is a highly significant preference for transitions of cytosine to occur at the first nucleotide position and for transitions of guanosine to occur at the second nucleotide position in the mutated codon (read 3' to 5'). Thus, the mechanisms involved appear to be sensitive to codon reading frames and to have an intrinsic ability to differentiate between the cytosines on the nontranscribed strand and those on the transcribed strand in the context of an open "transcription bubble."

Genesis of ancestral haplotypes: RNA modifications and reverse transcription-mediated polymorphisms.

Understanding the genesis of the block haplotype structure of the genome is a major challenge. With the completion of the sequencing of the Human Genome and the initiation of the HapMap project the concept that the chromosomes of the mammalian genome are a mosaic, or patchwork, of conserved extended block haplotype sequences is now accepted by the mainstream genomics research community. Ancestral Haplotypes (AHs) can be viewed as a recombined string of smaller Polymorphic Frozen Blocks (PFBs). How have such variant extended DNA sequence tracts emerged in evolution? Here the relevant literature on the problem is reviewed from various fields of molecular and cell biology particularly molecular immunology and comparative and functional genomics. Based on our synthesis we then advance a testable molecular and cellular model. A critical part of the analysis concerns the origin of the strand biased mutation signatures in the transcribed regions of the human and higher primate genome, A-to-G versus T-to-C (ratio ∼ 1.5 fold) and C-to-T versus G-to-A (≥ 1.5 fold). A comparison and evaluation of the current state of the fields of immunoglobulin Somatic Hypermutation (SHM) and Transcription-Coupled DNA Repair focused on how mutations in newly synthesized RNA might be copied back to DNA thus accounting for some of the genome-wide strand biases (e.g., the A-to-G vs T-to-C component of the strand biased spectrum). We hypothesize that the genesis of PFBs and extended AHs occurs during mutagenic episodes in evolution (e.g., retroviral infections) and that many of the critical DNA sequence diversifying events occur first at the RNA level, e.g., recombination between RNA strings resulting in tandem and dispersed RNA duplications (retroduplications), RNA mutations via adenosine-to-inosine pre-mRNA editing events as well as error prone RNA synthesis. These are then copied back into DNA by a cellular reverse transcription process (also likely to be error-prone) that we have called "reverse transcription-mediated long DNA conversion." Finally we suggest that all these activities and others can be envisaged as being brought physically under the umbrella of special sites in the nucleus involved in transcription known as "transcription factories."