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Background Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. Results By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a “hot spot” where one deletion breakpoint occurred within the region of 6–9 kb and another within 13–16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6–9 kb and 13–16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470–8482 bp (base pair) and a second arm at 13,447–13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Conclusions Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Metastatic relapse after treatment is the leading cause of cancer mortality, and known resistance mechanisms are missing for most treatments administered to patients. To bridge this gap, we analyze a pan-cancer cohort (META-PRISM) of 1,031 refractory metastatic tumors profiled via whole-exome and transcriptome sequencing. META-PRISM tumors, particularly prostate, bladder, and pancreatic types, displayed the most transformed genomes compared with primary untreated tumors. Standard-of-care resistance biomarkers were identified only in lung and colon cancers—9.6% of META-PRISM tumors, indicating that too few resistance mechanisms have received clinical validation. In contrast, we verified the enrichment of multiple investigational and hypothetical resistance mechanisms in treated compared with nontreated patients, thereby confirming their putative role in treatment resistance. Additionally, we demonstrated that molecular markers improve 6-month survival prediction, particularly in patients with advanced breast cancer. Our analysis establishes the utility of the META-PRISM cohort for investigating resistance mechanisms and performing predictive analyses in cancer.Significance:This study highlights the paucity of standard-of-care markers that explain treatment resistance and the promise of investigational and hypothetical markers awaiting further validation. It also demonstrates the utility of molecular profiling in advanced-stage cancers, particularly breast cancer, to improve the survival prediction and assess eligibility to phase I clinical trials.This article is highlighted in the In This Issue feature, p. 1027

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3’ nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

These figures provide complementary information for the figures presented in the main text and illustrations of the methods section.

These tables present clinical data, sample data, whole-exome and transcriptome sequencing results, literature references, and external data sources that were used for the analyses presented in the main text.

The process of domestication is associated with decrease in effective population size, which in turn leads to accumulation of slightly-deleterious mutations due to genetic drift. To maintain genome quality at a high level, we propose to use a stress-induced strong purifying selection, which based on negative epistasis, can effectively eliminate organisms with an excess of deleterious variants. Here, to identify stress factors, which interact with the effect of deleterious mutations we performed a proof-of-principle experiment with several regimes of a heat shock. We observed that fitness of mutated versus wild-type carp lines drops stronger after heat shock, which is a signature of a negative epistasis. Although the observed trend is promising, the effect of the epistasis is weak and unstable from family to family. Thus, more deep tuning of heat shock regimes is needed to uncover the most efficient combination of factors (absolute temperature, duration, stage of the embryo development) aggravating the burden of deleterious mutations and thus exposing them to the selection.

Heat shock proteins in parallel with their main and originally discovered function – maintenance of folded proteins under stressful conditions, can play also background buffering role – by folding proteins with an excess of slightly-deleterious nonsynonymous variants (SDNV). Here we tested several scenarios of this buffering role. On the comparative species scale, we demonstrated that low-Ne species are characterized by a higher expression level of hsp90 which can be explained by the excess of SDNV. On the comparative tissue level, we showed that long-lived tissues have also a higher hsp90 expression level, which can be advantageous to maintain the functionality of proteins. On the comparative gene level, we demonstrated that purifying selection of hsp90 in low-Ne-species did not relax as strongly as it happens for control genes, similar to hsp90. Additionally, we demonstrated that hsp clients versus non-clients are characterised by decreased level of selective constraints; demonstrate stronger relaxation of purifying selection in low-Ne species; have an excess of slightly-deleterious variants associated with complex disease phenotypes in humans; have an excess of pathological variants associated with clinical phenotypes in humans, suggesting that clients, being buffered by hsp90 can degenerate a bit more as compared to non-clients. Altogether, our results show that the secondary role of hsp, buffering of SDNV, is widespread and universal affecting properties of species, tissues and genes. A deep understanding of the buffering role of hsp90 will help to predict the deleterious effect of each variant in the human genome more precisely as well as will extend the application of the effectively-neutral theory of molecular evolution.

Using low-coverage, whole-genome sequences of trophectoderm biopsies from 11,610 human blastocyst-stage embryos, we analyzed the relationship between chromosomal abnormalities and mitochondrial (mt) DNA dynamics. Comparing 6,208 aneuploid and 5,402 euploid embryos in cohort studies, we found that mtDNA content in aneuploid embryos was significantly higher than that in euploid embryos. This outcome was confirmed through intrafamilial analyses of embryos with matched parents andin vitrofertilization cycles, and it occurred independent of maternal age. Additional human population-based studies uncovered a higher abundance of ultra-rare mtDNA variants located in never-altered positions in the human population in aneuploid compared to euploid embryos in both cohort- and family-based analyses. This maternal age-independent association of increased mtDNA content and aneuploidy in human embryos may reflect a novel mechanism of purifying selection against potentially deleterious mtDNA variants, which arise from germline or early developmental mtDNA damaging events, that occurs in human embryos prior to implantation.

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis (TLS) DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyzed 38 skin cancer genomes from five XP groups. We found that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments withPOLH-KO cell line revealed the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3’ nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

Evolution is a function of mutagenesis and selection. To analyse the role of mutagenesis on the structure of the SARS-CoV-2 genome, we reconstructed the mutational spectrum, which was highly C>U and G>U biased. This bias forces the SARS-CoV-2 genome to become increasingly U-rich unless selection cancels it. We analysed the consequences of this bias on the composition of the most neutral (four-fold degenerate synonymous substitutions) and the least neutral positions (nonsynonymous substitutions). The neutral nucleotide composition is already highly saturated by U and, according to our model, it is at equilibrium, suggesting that in the future, we don’t expect any more increase in U. However, nonsynonymous changes continue slowly evolve towards equilibrium substituting CG-rich amino-acids (“losers”) with U-rich ones (“gainers”). This process is universal for all genes of SARS-CoV-2 as well as for other coronaviridae species. In line with the direction mutation pressure hypothesis, we show that viral-specific amino acid content is associated with the viral-specific mutational spectrum due to the accumulation of effectively neutral slightly deleterious variants (losers to gainers) during the molecular evolution. The tuning of a protein space by the mutational process is expected to be typical for species with relaxed purifying selection, suggesting that the purging of slightly-deleterious variants in the SARS-CoV-2 population is not very effective, probably due to the fast expansion of the viral population during the pandemic. Understanding the mutational process can help to design more robust vaccines, based on gainer-rich motifs, close to the mutation-selection equilibrium.

The hypothesis that the evolution of humans involves hybridization between diverged species has been actively debated in recent years. We present the following novel evidence in support of this hypothesis: the analysis of nuclear pseudogenes of mtDNA (“NUMTs”). NUMTs are considered “mtDNA fossils” as they preserve sequences of ancient mtDNA and thus carry unique information about ancestral populations. Our comparison of a NUMT sequence shared by humans, chimpanzees, and gorillas with their mtDNAs implies that, around the time of divergence between humans and chimpanzees, our evolutionary history involved the interbreeding of individuals whose mtDNA had diverged as much as ~4.5 Myr prior. This large divergence suggests a distant interspecies hybridization. Additionally, analysis of two other NUMTs suggests that such events occur repeatedly. Our findings suggest a complex pattern of speciation in primate/human ancestors and provide one potential explanation for the mosaic nature of fossil morphology found at the emergence of the hominin lineage. A preliminary version of this manuscript was uploaded to the preprint server BioRxiv in 2017 (10.1101/134502).

Metastatic relapse after treatment is the primary cause of cancer morbidity and mortality. While genetic mechanisms of primary tumors and, to a lesser extent, metastatic cancers have been studied in large cohorts, refractory metastatic tumors are not yet sufficiently characterized. Markers of aggressiveness and resistance that molecular profiling can extract from these tumors have yet to be identified and incorporated into clinical care. In this study, we present a pan-cancer cohort of 1,031 metastatic tumors (which we refer to as META-PRISM) that are resistant to at least one systemic therapy or with no approved treatment options. We retrieved the complete clinical history of patients and performed whole-exome (n=571) and transcriptome sequencing (n=947) for this cohort. The prevalence of detected cancer biomarkers was assessed and compared to an external cohort of primary tumors. In the META-PRISM cohort, we observed an increase in (i) whole-genome duplication frequency, (ii) tumor mutational burden, (iii) germline cancer-predisposing variants, and (iv) somatic alterations in cancer genes, including KRAS, EGFR, CCND1, MYC, and TP53, as compared to the tumor type-matched primary tumors. The most extensive increase in genomic variation at metastatic stage was observed in prostate bladder, and pancreatic cancer types. We also identified enrichment of standard-of-care resistance biomarkers in most cancer types. However, only 9.3% of tumors harbored at least one such biomarker, indicating that the current understanding of resistance mechanisms remains insufficient. Our cohort demonstrated a significantly improved 6-month survival prediction from models incorporating molecular markers over models with only clinical markers for breast cancer patients and to a lesser extent for other studied tumor types. Overall, our data establish a unique resource for investigating treatment resistance mechanisms and performing predictive analyses in cancer. Citation Format: Yoann Pradat, Julien Viot, Konstantin Gunbin, Andrey Yurchenko, Luigi Cerbone, Marc Deloger, Guillaume Grisay, Loic Verlingue, Véronique Scott, Ismael Padioleau, Leonardo Panunzi, Stefan Michiels, Antoine Hollebecque, Gerome Jules-Clément, Laura Mezquita, Antoine Lainé, Yohann Loriot, Benjamin Besse, Luc Friboulet, Fabrice André, Paul-Henry Cournède, Daniel Gautheret, Sergey Nikolaev. Integrative pan-cancer genomic and transcriptomic analyses of refractory metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6570.

Metastatic relapse after treatment is the primary cause of cancer morbidity and mortality. While genetic mechanisms of primary tumors and, to a lesser extent, metastatic cancers have been studied in large cohorts, refractory metastatic tumors are not yet sufficiently characterized. Markers of aggressiveness and resistance that molecular profiling can extract from these tumors have yet to be identified and incorporated into clinical care. In this study, we present a pan-cancer cohort of 1,031 metastatic tumors (which we refer to as META-PRISM) that are resistant to at least one systemic therapy or with no approved treatment options. We retrieved the complete clinical history of patients and performed whole-exome (n=571) and transcriptome sequencing (n=947) for this cohort. The prevalence of detected cancer biomarkers was assessed and compared to an external cohort of primary tumors. In the META-PRISM cohort, we observed an increase in (i) whole-genome duplication frequency, (ii) tumor mutational burden, (iii) germline cancer-predisposing variants, and (iv) somatic alterations in cancer genes, including KRAS, EGFR, CCND1, MYC, and TP53, as compared to the tumor type-matched primary tumors. The most extensive increase in genomic variation at metastatic stage was observed in prostate cancer. We also identified enrichment of standard-of-care resistance biomarkers in most cancer types. However, only 7.6% of tumors harbored at least one such biomarker, indicating that the current understanding of resistance mechanisms remains insufficient. Our cohort demonstrated a significantly improved 6-month survival prediction from models incorporating molecular markers over models with only clinical markers for breast cancer patients and to a lesser extent for other studied tumor types. Overall, our data establish a unique resource for investigating treatment resistance mechanisms and performing predictive analyses in cancer. Citation Format: Yoann Pradat, Julien Viot, Konstantin Gunbin, Andrei Iurchenko, Marc Deloger, Luigi Cerbone, Guillaume Grisay, Loic Verlingue, Veronique Scott, Stefan Michiels, Antoine Hollebecque, Gerome Jules-Clement, Antoine Laine, Luc Friboulet, Laura Mezquita, Yohann Loriot, Benjamin Besse, Fabrice Andre, Paul-Henry Cournede, Daniel Gautheret, Sergey Nikolaev. Integrative pan-cancer genomic and transcriptomic analyses of refractory metastatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR009.

Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Deletions in mitochondrial DNA (mtDNA) are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded DNA might play a role in the formation of deletions. By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9kb and another within 13-16kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp and a second arm at 13447-13459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. An analysis of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Mutational spectrum of the mitochondrial genome (mtDNA) does not resemble signatures of any known mutagens and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra may be affected by the oxidative damage which is increasing with cellular and organismal aging. However, the well-documented mutational signature of the oxidative damage, G>T substitutions, is typical only for the nuclear genome while it is extremely rare and age-independent in mtDNA. Thus it is still unclear if there is a mitochondria - specific mutational signature of the oxidative damage. Here, reconstructing mtDNA mutational spectra for human cancers originated from 21 tissues with various cell turnover rate, human oocytes fertilized at different ages, and 424 mammalian species with variable generation length which is a proxy for oocyte age, we observed that the frequency of AH>GHsubstitutions (H- heavy chain notation) is positively correlated with cellular and organismal longevity. Moreover, this mutational bias from AHto GHaffects nucleotide content at the fourfold degenerative synonymous positions leading to a deficit of AHand excess of GH, which is especially pronounced in long-lived mammals. Taking into account additionally, that AH>GHis sensitive to time being single stranded during mtDNA asynchronous replication and A>G is associated with oxidative damage of single-stranded DNA in recent bacterial experiments we propose that AH>GHis a mutational signature of oxidative damage in mtDNA.

Introduction: Increasingly, the emergence and evolution of our species is being tied to genetic exchange between divergent lineages within ~1Ma (e.g., Neanderthals, Denisovans). However, little is known about genetic exchange during earlier (pre-1Ma) human evolution and between more divergent lineages. Results: We present evidence of hybridization within human lineage, show that it likely happened between highly divergent (~4.5My) lineages, more than once. We use analysis of nuclear pseudogenes of mtDNA (“NUMTs”). NUMTs are considered “mtDNA fossils”, as they preserve sequences of ancient mtDNA because mutational rate in the nucleus is much lower than in mtDNA. We demonstrate that a NUMT on human chromosome 5, which is shared by chimpanzee and gorilla, had descended from a mitochondrial genome that had been divergent from our ancestor’s mtDNA by ~4.5% at the time of pseudogene insertion. This implies that this pseudogene should have been inserted in a hominid that at that time had been diverged by about 4.5My of evolution from the hominid that at that time carried our mtDNA lineage. In order for this pseudogene and our mtDNA to end up in the same body, these two hominids should have mated with each other. The large divergence implies a distant interspecies (or even inter-generic) hybridization. Additionally, analysis of two other NUMTs (on Chr11 and Chr7) suggests that hybridization events occurred repeatedly. To exclude the large ancestral population size effect we show that mtDNA divergence in extant ape populations does not depend on population size. Discussion: It is thought that within mammals, it takes ~2-4My to establish reproductive isolation. However, fertile inter-generic hybrids have been documented among several primates, separated by ca. 4My. Very recently, hybridization between Colobine genera separated by ~5 My was reported to involve a NUMT scenario similar to what we had proposed human ancestors. Interestingly, phylogenic analysis consistently places the chr5 NUMT insertion around the time of the Homo/Pan split. Intriguingly, certain hominin fossils of that epoch have been interpreted alternately as more human-like or more ape-like. Such morphological mosaicisity could potentially be explained by hybridization. Fixation of NUMTs in question within population should have been rather efficient, since these pseudogenes appear to have been fixed in more than one population. Thus their spread across populations might have been driven by selection. Indeed, NUMTs on chr5 and chr11 are located in 3’ regions of functional genes. Most intriguingly, Ps11 is located 3’ to the RNF141/ZNF230 gene, essential for spermatogenesis. NUMT might have served as an expression modifier for RNF141, resulting in reproductive advantage. Indeed, RNF141 demonstrates selectively driven expression shift in testis of the ancestor of hominines.