Biology subjects

Fleischmann, Z.

Publications and source records attributed to Fleischmann, Z..

10 recordsLinked to original sources

bioRxiv2026

Landscape of reversion alterations in homologous recombination genes reveals evolutionary constraints

Reversion mutations (REVs) restore homologous recombination repair (HRR) and confer resistance to PARP inhibitors (PARPi) in HR-deficient cancers. Yet, their prevalence, mechanisms, and biological constraints remain undefined. We analyzed genomic profiling of 609,464 tissue and liquid biopsy samples across multiple cancer types to delineate the pan-cancer landscape of REVs. REVs were identified in eight HRR genes, most frequently BRCA2 and BRCA1 and notably never in ATM or CHEK2. REVs exclusively impacted truncating pathogenic variants, predominantly through large in-frame and exon-level deletions, associated with repetitive sequences. Conserved functional domains are relatively depleted of REVs. Structural modeling and functional studies support that exon-level deletions preserve critical domain architecture and confer PARPi resistance. The study establishes HRR reversion as a structurally permissive, yet evolutionarily constrained, resistance mechanism with implications on response, monitoring, and therapeutic strategy. One Sentence SummaryHomologous recombination reversion evolves through structurally tolerated, microhomology-driven alterations that restore DNA repair under therapeutic selection across cancers.

cancer biology↗

bioRxiv2026

Positive germline selection of mtDNA mutations: evidence from the oocyte.

Purifying selection is the removal of detrimental mutations. Nuclear DNA mutations are purged by removing mutant individuals, embryos, or germline cells. Because mtDNA is present in numerous copies per cell, purifying selection of mtDNA mutations introduces an additional layer of complexity. The intracellular mutant fraction can change due to clonal expansion/reduction of mutant mtDNA molecules in germline cells. Different research groups have reported either negative (purifying), or positive (destructive) germline mtDNA selection. In this study, we use recently published high-fidelity data on mtDNA mutations in individual human oocytes from the Makova laboratory (Arbeithuber et al., 2025) to resolve this disagreement. To assess selection, we used the "selective expansion" metric St, which compares the average weighted rate of clonal expansion in a set of mutations tested for selection (e.g. non-synonymous mutations) to that of the set of synonymous mutations. We found that, on average, noncoding oocyte mutations in the control region expand faster than synonymous mutations, i.e., are under positive selection. Intriguingly, mutations in the coding region, the detrimental ones in particular, were also on average under positive selection. The prescence of average positive selection does not preclude purifying selection against individual mutations; it only indicates that positive selection dominates in aggregate mutational dynamics. Positive selection impacts the dynamics of de novo germline mutations that arise in primordial germ cells or oocytes and have not yet been inherited by the next generation. Contrastly, mutations that are passed to subsequent generations are under average purifying selection. We suggest that initial positive selection begets subsequent purifying selection by increasing intracellular mutant fractions to levels at which their deleterious effects become phenotypically apparent and can be efficiently removed.

genetics↗

bioRxiv2023

A novel, wave-shaped profile of germline selection of pathogenic mtDNA mutations is discovered by bypassing a classical statistical bias.

The shift of the level of disease-causing mtDNA mutations (heteroplasmy) from mother to child is typically negatively correlated with the mothers heteroplasmy (Hm). In other words, mothers with low Hm tend to have children with a higher mutation level (Hch) than their own. In contrast, mothers with high Hm typically see a decrease in heteroplasmy in their children. This trend has been commonly interpreted as a result of a descending germline selection profile, i.e., positive selection at low Hm, gradually turning negative at high Hm. Here we demonstrate, however, that the negative correlation is mostly driven by RTM, or Regression To the Mean, a classical statistical bias. We further show that RTM can be nullified by using the average between the mothers and childs heteroplasmy, as a new variable, instead of the commonly used mothers heteroplasmy in blood. Additionally, we demonstrate that mother/child average is a better approximation of the actual germline heteroplasmy. Moreover, the elimination of RTM revealed a previously hidden wave-shaped HS-profile (positive mother-to-child shift at intermediate average mother-child heteroplasmy, decreasing towards high and low average heteroplasmy). In confirmation of this finding, we show that simulations that involve both wave-shaped HS-profile and RTM, reproduce the observed patterns of inheritance of mtDNA mutations in unprecedented detail. From the health care perspective, the uncovering of the wave-shaped HS-profile (and the removal of the RTM bias) are crucial for families affected by mtDNA disease. From the fundamental perspective, the wave- shaped profile offers a novel understanding of the dynamics of mtDNA in the germline and a novel potential mechanism that prevents the spread of detrimental mtDNA mutations in the population. SignificanceFrom the clinical perspective, the existence of wave-shaped selection may improve predictions and decisions for families affected by mtDNA diseases. From the fundamental perspective, it provides insight into the dynamics of general mtDNA mutations in the germline and in the population, as long as they follow wave-shaped selection profile. In Fig. 1, blue and red arrows represent the direction of expected changes of the heteroplasmy in a lineage with time/generations. With wave- shaped selection (Fig. 1B), a great majority of nascent low- fraction mutations are expected to converge back to zero and vanish. However, due to random intracellular genetic drift, some mutations will, occasionally, expand and enter the range of positive selection. Then they will be expanded by the selection to higher, detrimental levels, and become prone to downstream removal via death of highly mutated germ cells or inability of highly sick individuals to continue their lineage. In this way, the wave-shaped selection may help to prevent the spread of detrimental mutations in the population and in the species. In contrast, if the descending selection profile (Fig. 1A) was in effect, the nascent low heteroplasmy detrimental mutations would have been pushed to intermediate heteroplasmy levels where they will stay longer in hidden disease carriers enabling effective spread of mutation in the population. O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/568140v2_fig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1142cc0org.highwire.dtl.DTLVardef@186002aorg.highwire.dtl.DTLVardef@74d176org.highwire.dtl.DTLVardef@163ca6c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO (Graphic Summary) Pathogenic mtDNA mutations that cause a host of devastating inherited diseases are usually thought to follow an intriguing inheritance trend: Mothers with low levels of mutation (called mother heteroplasmy, Hm) tend to bear children with higher child heteroplasmy (Hch) then their own which constitutes positive Heteroplasmy Shift (HS=Hch-Hm). In contrast, mothers with high heteroplasmy Hm bear children with lower heteroplasmy Hc (negative HS). C_FIG

genomics↗

bioRxiv2022

Reanalysis of mtDNA mutations of human primordial germ cells (PGCs) reveals significant contamination with NUMTs, and challenges predominantly purifying selection in late PGCs

The resilience of the mitochondrial genome (mtDNA) to a high mutational pressure depends, in part, on negative purifying selection in the germline. A paradigm in the field has been that such selection, at least in part, takes place in primordial germ cells (PGCs). Specifically, Floros et al. (Nature Cell Biology 20: 144-51) reported an increase in the synonymity of mtDNA mutations (a sign of purifying selection) between pooled early-stage and late-stage PGCs. We re-analyzed Floros et al. pooled PGC data and noticed that their mutational dataset was significantly contaminated with single nucleotide variants (SNVs) derived from a nuclear sequence of mtDNA origin (NUMT) located on chromosome 5. Contamination was caused by co-amplification of the NUMT sequence by cross-specific PCR primers. Importantly, when we removed NUMT-derived SNVs, the evidence of purifying selection was abolished. In addition to pooled PGCs, Floros et al. reported the analysis of single late-stage PGCs, which were amplified with different sets of PCR primers that cannot amplify the NUMT sequence. Accordingly, we found no NUMT-derived SNVs among single PGCs mutations. Interestingly, single PGC mutations show a decrease of synonymity with increased intracellular mutant fraction. This pattern is incompatible with predominantly negative selection. This suggests that germline selection of mtDNA mutations is a complex phenomenon and that the part of this process that takes place in PGCs may be predominantly positive. However counterintuitive, positive germline selection of detrimental mtDNA mutations has been reported previously and potentially may be evolutionarily advantageous.

genomics↗

bioRxiv2022

The butterfly effect: mutational bias of SARS-CoV-2 affects its pattern of molecular evolution on synonymous and nonsynonymous levels

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 dont 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.

evolutionary biology↗

bioRxiv2022

Integrative analysis of a large real-world cohort of small cell lung cancer identifies distinct genetic subtypes and insights into histological transformation

Small cell lung cancer (SCLC) is a recalcitrant neuroendocrine carcinoma with dismal survival outcomes. A major barrier in the field has been the relative paucity of human tumors studied. Here we provide an integrated analysis of 3,600 "real-world" SCLC cases. This large cohort allowed us to identify new recurrent alterations and new genetic subtypes, including STK11-mutant tumors (1.7%) and TP53/RB1 wild-type tumors (5.5%), of which 12.7% were human papillomavirus-positive. In our cohort, gene amplifications on 4q12 are associated with increased overall survival while CCNE1 amplification is associated with decreased overall survival. We also identify more frequent alterations in the PTEN pathway in brain metastases. Finally, profiling cases of SCLC containing oncogenic drivers typically associated with NSCLC demonstrates that SCLC transformation may occur across multiple distinct molecular cohorts of NSCLC. These novel and unsuspected genetic features of SCLC may help personalize treatment approaches for this fatal form of cancer. STATEMENT OF SIGNIFICANCEMinimal changes in therapy and survival outcomes have occurred in SCLC for the past four decades. The identification of new genetic subtypes, novel recurrent mutations, and an improved understanding of the mechanisms of transformation to SCLC from NSCLC may guide the development of personalized therapies for subsets of patients with SCLC.

cancer biology↗

bioRxiv2021

Nuclear DNA-encoded fragments of mitochondrial DNA (mtDNA) confound analysis of selection of mtDNA mutations in human primordial germ cells.

The resilience of the mitochondrial genome to a high mutational pressure depends, in part, on purifying selection against detrimental mutations in the germline. It is crucial to understand the mechanisms of this process. Recently, Floros et al. concluded that much of the purifying selection takes place during the proliferation of primordial germ cells (PGCs) because, according to their analysis, the synonymity of mutations in late PGCs was seemingly increased compared to those in early PGCs. We re-analyzed the Floros et al. mutational data and discovered a high proportion of sequence variants that are not true mutations, but originate from NUMTs, the latter of which are segments of mitochondrial DNA (mtDNA) inserted into nuclear DNA, up to millions of years ago. This is a well-known artifact in mtDNA mutational analysis. Removal of these artifacts from the Floros et al. dataset abolishes the reported effect of purifying selection in PGCs. We therefore conclude that the mechanism of germline selection of mtDNA mutations remains open for debate, and more research is needed to fully elucidate the timing and nature of this process.

genomics↗

bioRxiv2020

A mitochondrial mutational signature of temperature and longevity in ectothermic and endothermic vertebrates.

The variation in the mutational spectrum of the mitochondrial genome (mtDNA) among species is not well understood. Recently, we demonstrated an increase in A>G substitutions on a heavy chain (hereafter AH>GH) of mtDNA in aged mammals, interpreting it as a hallmark of age-related oxidative damage. In this study, we hypothesized that the occurrence of AH>GH substitutions may depend on the level of aerobic metabolism, which can be inferred from an organisms body temperature. To test this hypothesis, we used body temperature in endotherms and environmental temperature in ectotherms as proxies for metabolic rate and reconstructed mtDNA mutational spectra for 1350 vertebrate species. Our results showed that temperature was associated with increased rates of AH>GH and asymmetry of AH>GH in different species of ray-finned fishes and within geographically distinct clades of European anchovy. Analysis of nucleotide composition in the most neutral synonymous sites of fishes revealed that warm-water species were expectedly more A-poor and G-rich compared to cold-water species. Finally, we extended our analyses to all vertebrates and observed higher AH>GH and increased asymmetry of AH>GH in warm-blooded (mammals and birds) compared to cold-blooded (Actinopterygii, amphibia, reptilia) vertebrate classes. Overall, our findings suggest that temperature, through its influence on metabolism and oxidative damage, shapes the mutational properties and nucleotide content of the mtDNA in all vertebrates.

evolutionary biology↗

bioRxiv2019

Quasi-Mendelian Paternal Inheritance of mitochondrial DNA: A notorious artifact, or anticipated mtDNA behavior?

A recent report by Luo et al (2018) in PNAS (DOI:10.1073/pnas.1810946115) presented evidence of biparental inheritance of mitochondrial DNA. The pattern of inheritance, however, resembled that of a nuclear gene. The authors explained this peculiarity with Mendelian segregation of a faulty gatekeeper gene that permits survival of paternal mtDNA in the oocyte. Three other groups (Vissing, 2019; Lutz-Bonengel and Parson, 2019; Salas et al, 2019), however, posited the observation was an artifact of inheritance of mtDNA nuclear pseudogenes (NUMTs), present in the fathers nuclear genome. We present justification that both interpretations are incorrect, but that the original authors did, in fact, observe biparental inheritance of mtDNA. Our alternative model assumes that because of initially low paternal mtDNA copy number these copies are randomly partitioned into nascent cell lineages. The paternal mtDNA haplotype must have a selective advantage, so seeded cells will tend to proceed to fixation of the paternal haplotype in the course of development. We use modeling to emulate the dynamics of paternal genomes and predict their mode of inheritance and distribution in somatic tissue. The resulting offspring is a mosaic of cells that are purely maternal or purely paternal - including in the germline. This mosaicism explains the quasi-Mendelian segregation of the paternal mDNA. Our model is based on known aspects of mtDNA biology and explains all of the experimental observations outlined in Luo et. al., including maternal inheritance of the grand-paternal mtDNA.

genetics↗

bioRxiv2019

Mitochondrial mutational spectrum provides an universal marker of cellular and organismal longevity

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>GH substitutions (H - heavy chain notation) is positively correlated with cellular and organismal longevity. Moreover, this mutational bias from AH to GH affects nucleotide content at the fourfold degenerative synonymous positions leading to a deficit of AH and excess of GH, which is especially pronounced in long-lived mammals. Taking into account additionally, that AH>GH is 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>GH is a mutational signature of oxidative damage in mtDNA.

genomics↗