Inflammation-induced mutagenesis Petr Müller Molecular and Cellular Pathophysiology Mutations By effect on structure mutation is an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA Large scale mutations (Chromosomal abnormalities) Small-scale mutations • Deletion • Duplication, amplification • Inversion • Translocation • Insertion • Loss of heterogisosity • Aneuploidity • Insertions • Deletions • Substitution mutations / point mutations • Missense • Nonsense • Silent Spontaneous vs induced mutations Tautomerism Depurination Deamination Slipped strand mispairing Replication slippage DNA replication Mitosis -chromosome segragation Chromosome abnormality Amplification Translocation Aneuploidity DNA repair -> genomic instability Double strand break repair BRCA1, BRCA2 CHK2 ATM Mismatch repair (MMR) MLH1, MLH2, MLH3, PMS1, and PMS2 Nucleotide excision repair (NER) Xeroderma pigmentosum (XPC,..) DNA polymerase POLD, POLE Transition • mutation that changes a purine nucleotide to another purine (A ↔ G), or a pyrimidine nucleotide to another pyrimidine (C ↔ T). • Approximately two out of three single nucleotide polymorphisms (SNPs) are transitions. • Transitions can be caused by oxidative deamination and tautomerization. Point mutations Transversion • mutation in DNA in which a single (two ring) purine (A or G) is changed for a (one ring) pyrimidine (T or C), or vice versa. • A transversion can be spontaneous, or it can be caused by ionizing radiation or alkylating agents. Mutations by impact on protein sequence Coding region: • Point mutations • Missense • Nonsense • Silent • Frameshift mutations • Insertions • Deletions • (Indels) • Genetic variation is an important force in evolution as it allows natural selection to increase or decrease frequency of alleles already in the population. • Genetic variation can be caused by mutation (which can create entirely new alleles in a population), random mating, random fertilization, and recombination between homologous chromosomes during meiosis (which reshuffles alleles within an organism’s offspring). • Genetic variation is advantageous to a population because it enables some individuals to adapt to the environment while maintaining the survival of the population. Bad and good mutations Adaptive immunity and controled mutagenesis IGG1 IGG2A Antibody isotypes Antibody genetics IGH locus chr. 14 44V x 27D x 6J IGL kappa (κ) locus chr. 2 IGL lambda (λ) locus chr. 22 Susumu Tonegawa Nobel prize 1987 V(D)J recombination Somatic hypermutation and affinity maturation Class switching V(D)J recombinations in lymphocyte differentiation H1 H2 H3 N C CDR1 CDR2 CDR3 VDJ recombination Class switching μ - IgM δ - IgD γ3 - IgG3 γ1 - IgG1 pseudogene α1 - IgA1 γ2 - IgG2 γ4 - IgG4 ε - IgE α2 - IgA2 S-region DSB NHEJ AID AP UDG Activation-Induced (Cytidine) Deaminase (AID), Uracil DNA glycosylase Apyrimidic/apurinic (AP)-endonucleases Tumor suppressor p53 - the guardian of genome David Lane • Most frequently mutated gene in cancer • Transcription factor • Germline mutation cause Li-Fraumeni syndrome • Tumor suppressor • Both alleles lost in cancer – (lost of heterozygosity) LOH Tumor suppressor p53 - the guardian of genome CGG -->Arg p53 mutational spectrum CpG island Genomic instability „Spontaneous“ mutations (aging and inflamation) CpG island Exogenous mutagens • Smoking • UV light • Alkylating agents • aflatoxin Mutation pattern Mutational signatures NGS Next Generation Sequencing TruSight™ Oncology 500 https://cancer.sanger.ac.uk/signatures/ Mutational Signatures Endogenous mutational process initiated by spontaneous deamination of 5-methylcytosine Mutational signatures Genomic instability „Spontaneous“ mutations (aging and inflamation) CpG island Exogenous mutagens • Smoking • UV light • Alkylating agents • aflatoxin Mutation pattern Mutational signatures POLE P286R Cells are able to actively induce mutations APOBEC family members APOBEC1 APOBEC2 APOBEC3A APOBEC3B APOBEC3C APOBEC3D APOBEC3F APOBEC3G APOBEC3H APOBEC4 AID (activation induced deaminase) • APOBEC ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like") is a family of evolutionarily conserved cytidine deaminases. • Discovered due to their ability to eliminate HIV infection • When misregulated, are a major source of mutation in numerous cancer types. • AID is a part of adaptive immunity; it is responsible for hypermutation of variable immunoglobulin regions in lymphocytes Cells are able to actively induce mutations The effects of mutations induced by the activity of APOBEC3 The role of APOBEC3 Mechanisms of innate immunity (fast but non-specific response) Detection of pathogenic microorganisms • Membrane receptors • Intracellular receptors of foreign nucleic acids • Cytokine signalling Intracellular signalling pathways Activation of transcription / gene expression • Expression of cytokines • Activation of specific immune response • Elimination of microorganisms • Use of gene Mechanisms of innate immunity Toll-like receptors (TLRs) TLR TLR NF-κB Activated by extracellular signals from pathogens: Polysacharides, RNA, DNA, proteins, lipopeptides, endotoxins,.. IRFs IFN IFNAR gamma-activated sequences, IFN-stimulated response elements GAS ISRE STAT Cyclic GMP-AMP synthase Cytoplasmic sensor of DNA Cyclic guanosine monophosphate– adenosine monophosphate cGAS ATP+GTPER Stimulator of interferon genes STING cGAMP Interferone receptors IRGs – interferone regulated genes Cytokines Host-directed editing enzymes: APOBEC, ADAR, AID.. Mechanism of action: Deamination of adenosine to inosine Destabilize RNA Mismatch pairing when replicated ADAR - adenosine deaminase acting on RNA responsible for binding to double stranded RNA (dsRNA) and converting adenosine (A) to inosine (I) by deamination. ADAR protein is a RNA-binding protein, which functions in RNA-editing through post-transcriptional modification of mRNA transcripts by changing the nucleotide content of the RNA Dysregulation associated with: Aicardi–Goutières syndrome and Bilateral Striatal Necrosis/Dystonia, cancer (HCC) The results suggest that the heterogeneous mutation patterns are mainly reflections of host (i) antiviral mechanisms that are achieved through APOBEC, ADAR, and ZAP proteins, and (ii) probable adaptation against reactive oxygen species. Signatures of Mutational Processes in Human CancerSignature 2 has been attributed to activity of the AID/APOBEC family of cytidine deaminases. Loss of genes in NF-κB signalling pathway Expansion of the APOBEC3 gene locus