Hall NE, Mamrot J, Frampton CMA, Read P, Steele EJ, Bischof RJ, Lindley RA (2018) Deaminase associated single nucleotide variants in blood and saliva-derived exomes from healthy subjects. BMC Medical Genomics (Submitted and under review: copy available on request).
Lindley RA (2018) Deaminase Targeted Somatic Mutation (TSM) Signatures: A Review Targeted Codon-Context DNA and RNA Deamination Mutations in Cancer Genome Diagnosis and Prognosis. Mutation Research (Submitted and under review: copy available on request).
Recent developments in somatic mutation analyses in cancer have led to the discovery of codon-context targeted somatic mutation signatures (TSM) in all cancer genomes examined thus far. As this research provides some novel insights into the origin of the majority of somatic point mutations in cancer genomes, it requires clear explanation for both the molecular and cellular scientist as well as cancer specialists in the clinic. This review will cover the background to TSM that arose from basic research into the mechanism of antigen-driven somatic hypermutation of immunoglobulin variable genes (Ig SHM), and how it has now been established that an Ig-SHM-like signature is written all over the cancer exome. Most importantly, the data now show that Ig-SHM-like cancer-associated signatures are highly targeted to Cytosine (C) and Adenosine (A) nucleotides in a characteristic codon-context fashion. The TSM related data and plausible mechanisms for the origin of these mutation signatures, based on the 3-D geometry of Transcription Bubbles, are discussed in depth. This review also provides a plausible framework for furthering our molecular understanding of how the established cytosine and adenosine deaminase mutators gain access to their respective motif defined DNA (C-sites) and RNA (A-sites) mutational targets within stalled Transcription Bubbles during gene expression facilitating cancer progression. It also highlights the clinical utility of these discoveries for the development of new genomic diagnostics and drug discovery for pre-cancerous and full-blown cancer patients.
Mamrot J, Balachandran S, Steele EJ, Lindley, RA (2018) Molecular model linking polarized Tumor Associated Macrophages (TAMs) with deaminase-mediated cancer progression mutation signatures. Scandinavian Journal of Immunology (Submitted and under review: copy available on request).
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 tumor associated macrophages (TAMs) is 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 a/b and g/d of V(D)Js of T Cell Receptors) required for pathogen antigen recognition by B-cells and T-cells respectively. We also discuss the possibility that extracellular vesicles (EVs) emanating from tumor 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.
Lindley RA (2018) A New Treaty Between Disease and Evolution: Are deaminases the “universal mutators” responsible for our own evolution? Published December 10, 2018 (Online First) in Edge Science, Vol 36, pp16-20.
As we enter a new evidence-based era driven by Next Generation Sequencing (NGS), a revolution in our understanding about the source of mutations and their association with disease is overwhelming the traditional neo-Darwinian view of evolution based on the idea of “random” mutations. The new “big data” algorithms being developed in this “Age of Google” are forcing us to fundamentally alter our assumptions about genes, disease, and how genetic variability is generated. The recently discovered genetic mechanisms giving rise to mutations and disease depend on environmentally driven and dynamic epigenetic gene regulation. These are coupled to highly targeted mutator mechanisms. This article describes the latest understanding of how these genetic mechanisms work together to pave a path to new evolutionary thought.
Lindley RA and Steele EJ (2018) ADAR and APOBEC editing signatures in viral RNA during acute-phase Innate Immune responses of the host-parasite relationship to Flaviviruses. Published May 13, 2018 (Online First) in Research Reports.
The origins of variability in positive single-stranded RNA Flaviviruses is poorly understood. Is the origin of the high rate of spontaneous mutations arising early in Flavivirus infections due solely to error-prone viral replication? Alternatively, are these mutations due to viral replicase incorporation of high frequency RNA deamination editing events? The data analyzed here strongly supports the second explanation. The viral genomes analysed include: a) acute phase Zika virus (ZIKV) genomes associated with microcephaly; b) hepatitis C virus (HCV) genomes from both acute and chronic infected hepatitis patients; and c) hepatitis B virus (HBV) genomes of patients infected from the early/acute phase of a large nosocomial outbreak. RNA mutations at motifs for APOBEC and ADAR deaminases were analyzed within the codon context structure, used previously for targeted somatic mutation (TSM) analyses of cancer exomes. The results show that transition mutations targeting MC3 nucleotide sites (ie. the third nucleotide position within the structure of the Mutated Codon read 5’ to 3’), and within known RNA editing motifs for APOBEC1, APOBEC3A and ADAR1/2 account for the majority of the mutations for ZIKV, HCV, as well as for HBV. The results also imply that drug therapy strategies might profitably focus on the RNA-based secondary structure of potential “druggable pockets” within apparently conserved regions of amino acid sequence within variant viral strains. Such sites could encode subtle changes in RNA secondary structures as potential vulnerable target regions in pathogenic variants. For ZIKV, such sites could include the hypermutable MC3-deaminase targeted sites.
Steele EJ and Lindley RA (2018) Germline V Repertoires: Origin, Maintenance, Diversification. Scandinavian Journal of Immunology, 87(6):e12670. DOI: 10.1111/sji.12670.
In our view Melvin Cohn1 has set out the logical guidelines towards a resolution of the very real enigma of the selectability of vertebrate germline Ig V repertoires under the current evolutionary paradigm …” A somatically derived repertoire scrambles this (germline VL + VH) substrate so that its specificities are lost, making it un-selectable in the germline. Consequently, evolution faced an incompatibility.” It is argued here in Reply that a reverse transcriptase-based soma-to-germline process (S->G) targeting germline V segment arrays goes some considerable way to resolving fundamental contradictions on the origin, maintenance and then real-time adaptive diversification of these limited sets of V segments encoded within various V repertoire arrays.
1. Cohn M. Somatic diversification of the B cell repertoire requires two cell subsets. Scand J Immunol. 2018;87:e12640. https://doi.org/10.1111/sji.12640
Lindley RA and Hall NE (2018) APOBEC and ADAR deaminases may cause many single nucleotide polymorphisms curated in the OMIM database. Mutation Research, 810:33-38. DOI: 10.1016/j.mrfmmm.2018.03.008
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. Our hypothesis is that many missense, nonsense and synonymous single nucleotide polymorphisms (SNPs) associated with clinically significant diseases arise by similar highly targeted deamination processes. The OMIM database was searched for SNPs associated with X-Linked Recessive Diseases. The nucleotide substitution patterns for 789 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, 37% were coincident with the dominant deamination motifs for AID, APOBEC3G, APOBEC3B, ADAR1/2. The results imply that a deamination of C-site and A-site targets have been written into the human germline for the chromosome wide exomic SNPs analysed. The TSM patterns reveal preferential targeting to known AID/APOBE and ADAR deaminase motifs at specific codon reading frame sites. This is consistent with previously observed mutation patterns arising in cancer genomes and hypermutated Ig genes during SHM implying that similar types of deaminase-mediated molecular processes occurring in somatic hypermutation and cancer are contributing causative drivers of human exomic SNPs.
Steele EJ and Lindley RA (2017) ADAR deaminase A-to-I editing of DNA and RNA moieties of RNA:DNA hybrids has implications for the mechanism of Ig somatic hypermutation. DNA Repair 55:1–6. DOI: 10.1016/j.dnarep.2017.04.004
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.
Steele EJ and Lindley RA (2017) Origin of High Affinity Germline V Elements? Online Comment on Sterner, E., et al 2017 Cell Reports 20:1681-1691. DOI: 10.1016/j.celrep.2017.07.050
Lindley RA, Humbert P, Larmer C, Akmeemana EH, Pendlebury CRR (2016) Association between Targeted Somatic Mutation (TSM) signatures and HGS-OvCa progression. Cancer Medicine. 5(9):2629-2640. DOI: 10.1002/cam4.825
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. Further, 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.
Steele EJ. (2016) Somatic hypermutation in immunity and cancer: Critical analysis of strand-biased and codon-context mutation signatures DNA Repair 45:1-24. DOI: 10.1016/j.dnarep.2016.07.001
For 30 years two general mechanisms have competed to explain somatic hypermutation of immunoglobulin (Ig) genes. The first, the DNA-based model, is focussed only on DNA substrates. The modern form is the Neuberger “DNA Deamination Model” based on activation-induced cytidine deaminase (AID) and short-patch error-prone DNA repair by DNA Polymerase-h operating around AID C-to-U lesions. The other is an RNA-based mechanism or the “Reverse Transcriptase Model” of SHM which produces strand-biased mutations at A:T and G:C base pairs. This involves error-prone cDNA synthesis via an RNA-dependent DNA polymerase copying the Ig pre-mRNA template and integrating the now error-filled cDNA copy back into the normal chromosomal site. The modern form of this mechanism depends on AID dC-to-dU lesions and long tract error-prone cDNA synthesis of the transcribed strand by DNA Polymerase-h acting as a reverse transcriptase. The evidence for and against each mechanism is critically evaluated. The conclusion is that all the SHM molecular data gathered since 1980 supports directly or indirectly the RNA/RT-based mechanism. All the data and critical analyses are systematically laid out so the reader can evaluate this conclusion for themselves.
Recently we have investigated whether similar RNA/RT-based mutator mechanisms explain how de novomutations arise in somatic tissues (cancer genomes). The data analyses indeed suggest that cancers arise via dysregulated “Ig-like SHM responses” involving rogue DNA and RNA deaminations coupled to genome-wide RT events. Further, Robyn Lindley has recently shown that the strand-biased mutations in cancer genome genes are also in “codon-context.” This has been termed Targeted Somatic Mutation (TSM) to highlight that mutations are far more targeted than previously thought in somatic tissues associated with disease. The TSM process implies an “in-frame DNA reader” whereby DNA and RNA deaminases at transcribed regions are guided in their mutagenic action, by the codon reading frame of the DNA.
Lindley RA (2013) The importance of codon context for understanding the Ig-like somatic hypermutation strand-biased patterns in TP53 mutations in breast cancer Codon structure flags TP53 mutation targets in breast cancer. Cancer Genetics. 5(9):2619-2640.
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 WAsites. 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.”
Lindley RA and Steele EJ (2013) Critical analysis of strand-biased somatic mutation signatures in TP53 versus Ig genes, in genome -wide data and the etiology of cancer. Review Article, ISRN Genomics. Vol 2013 Article ID 921418, 18 pages.
Previous analyses of rearranged immunoglobulin (Ig) variable genes (VDJs) concluded that the mechanism of Ig somatic hypermutation (SHM) involves the Ig pre-mRNA acting as a copying template resulting in characteristic strand biased somatic mutation patterns at A:T and G:C base pairs. We have since analysed cancer genome data and found the same mutation strandbiases,in toto or in part, in nonlymphoid cancers. Here we have analysed somatic mutations in a single well-characterised gene TP53. Our goal is to understand the genesis of the strand-biased mutation patterns in TP53—and in genome-wide data—that may arise by “endogenous” mechanisms as opposed to adduct-generated DNA-targeted strand-biased mutations caused by well characterised “external” carcinogenic influences in cigarette smoke, UV-light, and certain dietary components. The underlying strand-biased mutation signatures in TP53, for many non-lymphoid cancers, bear a striking resemblance to the Ig SHM pattern.A similar pattern can be found in genome-wide somatic mutations in cancer genomes that have also mutated TP53. The analysis implies a role for base-modified RNA template intermediates coupled to reverse transcription in the genesis of many cancers. Thus Ig SHM may be inappropriately activated in many non-lymphoid tissues via hormonal and/or inflammation-related processes leading to cancer.
Steele EJ, Lindley RA (2010) Somatic mutation patterns in non-lymphoid cancers resemble the strand biased somatic hypermutation spectra of antibody genes.Letter to the Editor, DNA Repair. 9:600-603.
It has been long accepted that many types of B cell cancer (lymphomas, myelomas, plasmacytomas, etc.) are derived from the antigen-stimulated B cell Germinal Center (GC) reaction , ,  and , i.e. they are aberrant products of the somatic hypermutation mechanism normally targeting rearranged immunoglobulin (Ig) variable genes (so-called V[D]J regions). Here we provide evidence that the somatic mutation patterns of some well-characterised cancer genomes  such as lung carcinomas, breast carcinomas and squamous cell carcinomas, strongly resemble in toto or in part the spectrum of somatic point mutations observed in normal physiological somatic hypermutation (SHM) in antibody variable genes . This implies that whilst SHM itself is a tightly regulated and beneficial mutational process for B lymphocytes of the immune system, aberrant mutations (or “crises”) or inadvertent activation of this complex activation-induced cytidine deaminase (AID)-dependent mechanism in a range of somatic tissue types could result, as often speculated , in cancer.
Steele EJ, Lindley RA, Wen J, Weiller GF (2006) Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots. DNA Repair 5:1346-1363.
Activation-induced cytidine deaminase (AID) initiates Phase I somatic hypermutation (SHM) of antibody genes by deaminating deoxy-cytosine to deoxy-uracil (C-to-U). These lesions trigger Phase II, a poorly understood process of error-prone repair targeting A-T pairs by DNA polymerase η (Pol η). Since Pol η is also a reverse transcriptase, Phase II could involve copying off RNA as well as DNA templates. We explore this idea further since in an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases (ADARs) are known to preferentially edit A nucleotides that are preceded by an A or U (W) in double-stranded RNA substrates. On this assumption and using a theoretical bioinformatics approach we show that a significant and specific correlation (P < 0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This implies roles for both RNA editing and reverse transcription during SHM in vivo and suggests definitive genetic experiments targeting the appropriate ADAR1 isoform (γINF-ADAR1) and/or Ig pre-mRNA templates.