Bogdan Efimenko

Mutational biases can influence genome composition, but their contribution to protein evolution remains difficult to quantify. Here we utilize a nearly neutral framework that translates nucleotide mutational spectra into expected amino acid substitution patterns and equilibrium amino acid compositions. Using SARS-CoV-2 as a model system, we show that the viral mutational spectrum explains more than 50% of the variation in observed single-nucleotide amino acid substitutions and predicts the overall direction of proteome-wide amino acid composition change during the COVID-19 pandemic. The predictive power of the model varies with selection regime: effectively neutral and weakly deleterious substitutions conform most closely to the mutational expectation, whereas strongly constrained sites and mutational hotspots show larger deviations. This indicates that departures from the nearly neutral baseline provide a quantitative proxy for purifying and positive selection. Extending the analysis across 34 RNA virus species, we find that positive-sense, negative-sense and double-stranded RNA viruses differ systematically in their mutational spectra, and that these differences are associated with predictable shifts in proteome composition. The same relationship is detectable in RNA-dependent RNA polymerase sequences from more than 77,000 viral species. These results indicate that taxon-specific mutational bias contributes persistently to protein evolution across evolutionary scales.

The mutational spectrum is an increasingly important molecular phenotype that quantitatively describes mutagenesis in a given gene and species, enabling future comparative analyses to reveal differences in underlying mutagenic processes, whether internal, such as DNA repair processes, or external, such as ecological niches and conditions. Mutation accumulation experiments, although time-consuming and costly, remain the standard approach for reconstructing bacterial neutral mutation spectra. Here, we present BacNeMu, a phylogenetically informed pipeline that reconstructs neutral mutational spectra of bacterial genomes using open databases GTDB, AnnoTree and KEGG Orthology, building on previously developed NeMu pipeline. BacNeMu reconstructs mutation spectra that closely match mutation accumulation experiments results while requiring substantially less time, enabling comparative analyses across diverse bacterial taxa. Applied to obligate aerobes and anaerobes, BacNeMu recovered the expected excess of T:A>C:G transitions, consistent with oxidative-damage-associated mutational patterns previously described in mitochondrial genomes and yeast single-strand. We further asked if any other ecologic factors influence a mutational spectrum. As a pilot we compared three species living under different temperatures: one strong thermophile - Thermotoga maritima, one psychrophile - Clostridium algidicarnis, and one with intermediate temperature tolerance - Psychrobacter sanguinis. In the thermophile, the relative frequency of T:A>C:G substitutions was higher than in the psychrophile, consistent with the hypothesis that GC-biased mutagenesis contributes to thermal adaptation, although C:G>T:A transitions predominate across all three species. BacNeMu provides a rapid, phylogenetically informed framework for generating biologically meaningful mutation spectra from open databases.

Objective: To explore the relationships between HPV genotypes, vaginal microbiota, oncogenic mutations, and cervical intraepithelial neoplasia (CIN) to develop a risk assessment model for predicting CIN. Methods: A cross‐sectional analysis was conducted on 264 women living in the Kaliningrad region, assessing CIN presence, HPV genotypes, vaginal microbiota composition, and mutations in key oncogenes. Results: HPV genotypes 16, 31, 33, 35, 58, and 66 were identified as the most prevalent among patients with HPV infections. However, in a multifactorial model, only HPV genotypes 16 and 58 demonstrated a significant association with high‐grade squamous intraepithelial lesions and above. Genetic analysis revealed mutations in TP53 and ERBB2 genes in 20 and 17 patients, respectively, with TP53 mutations showing a notable correlation with CIN progression. Two patients with diagnoses of “Negative for Intraepithelial Lesion or Malignancy” carried the TP53 R248W mutation, a well‐established neoplasia‐related variant, highlighting its potential as a predictor of precancerous conditions. Increased copy numbers of human DNA and Enterobacteriaceae DNA correlated with low‐grade squamous intraepithelial lesions, though many DNA‐derived features displayed instability in logistic regression models, suggesting the need for further validation. Conclusion: These findings suggest that although HPV genotypes, genetic mutations, and microbiota profiles may serve as markers for CIN, their predictive reliability requires further investigation. The present study represents the first large‐scale exploration of these factors conducted within the Russian female population.

Mitochondrial DNA (mtDNA) mutagenesis remains poorly understood despite its crucial role in disease, aging, and evolutionary tracing. In this study, we reconstructed a comprehensive 192-component mtDNA mutational spectrum for chordates by analyzing 118,397 synonymous mutations in the CytB gene across 1,697 species and five classes. This analysis revealed three primary forces shaping mtDNA mutagenesis: (i) symmetrical, replication-driven errors by mitochondrial polymerase (POLG), resulting in C > T and A > G mutations that are highly conserved across classes; (ii) asymmetrical, damage-driven C > T mutations on the single-stranded heavy strand with clock-like dynamics; and (iii) asymmetrical A > G mutations on the heavy strand, with dynamics suggesting sensitivity to oxidative damage. The third component, sensitive to oxidative damage, positions mtDNA mutagenesis as a promising marker for metabolic and physiological processes across various classes, species, organisms, tissues, and cells. The deconvolution of the mutational spectra into mutational signatures uncovered deficiencies in both base excision repair (BER) and mismatch repair (MMR) pathways. Further analysis of mutation hotspots, abasic sites, and mutational asymmetries underscores the critical role of single-stranded DNA damage (components ii and iii), which, uncorrected due to BER and MMR deficiencies, contributes roughly as many mutations as POLG-induced errors (component i).

The recognized importance of mutational spectra in molecular evolution is yet to be fully exploited beyond human cancer studies and model organisms. The wealth of intraspecific polymorphism data in the GenBank repository, covering a broad spectrum of genes and species, presents an untapped opportunity for detailed mutational spectrum analysis. Existing methods fall short by ignoring intermediate substitutions on the inner branches of phylogenetic trees and lacking the capability for cross-species mutational comparisons. To address these challenges, we present the NeMu pipeline, available at https://nemu-pipeline.com, a tool grounded in phylogenetic principles designed to provide comprehensive and scalable analysis of mutational spectra. Utilizing extensive sequence data from numerous available genome projects, NeMu rapidly and accurately reconstructs the neutral mutational spectrum. This tool, facilitating the reconstruction of gene- and species-specific mutational spectra, contributes to a deeper understanding of evolutionary mechanisms across the broad spectrum of known species.

One of the most important characteristics of each contemporary model of molecular evolution is the assumption that mutations occur in a constant manner; however, in the real world, the mutations are determined by the combination of the effects of DNA replication and repair. This affects the nucleotide composition of the genome and guides not just neutral but adaptive evolution ¹ . Mutation accumulation experiments are the de facto standard for the neutral mutation spectra estimation. However, recent studies have demonstrated that the mutation fraction under selection pressure is significantly underestimated in mutation accumulation experiments, and, therefore the precise extraction of neutral mutation spectra from mutation accumulation experiments is not trivial ² . To unravel the neutral mutation spectra, it is very important to analyze all the mutations available in depth, based on the evolutionary timescale, taking into consideration all the existing knowledge. In order to facilitate this analysis, we have created a novel pipeline, called NeMu ( https://biopipelines.kantiana.ru/nemu/ ).

To elucidate the primary factors shaping mitochondrial DNA (mtDNA) mutagenesis, we derived a comprehensive 192-component mtDNA mutational spectrum using 86,149 polymorphic synonymous mutations reconstructed from the CytB gene of 967 chordate species. The mtDNA spectrum analysis provided numerous findings on repair and mutation processes, breaking it down into three main signatures: (i) symmetrical, evenly distributed across both strands, mutations, induced by gamma DNA polymerase (about 50% of all mutations); (ii) asymmetrical, heavy-strand-specific, C>T mutations (about 30%); and (iii) asymmetrical, heavy-strand-specific A>G mutations, influenced by metabolic and age-specific factors (about 20%). We propose that both asymmetrical signatures are driven by single-strand specific damage coupled with inefficient base excision repair on the lagging (heavy) strand of mtDNA. Understanding the detailed mechanisms of this damage is crucial for developing strategies to reduce somatic mtDNA mutational load, which is vital for combating age-related diseases.

Molybdenum cofactor deficiency type B (MOCODB; #252160) is an autosomal recessive metabolic disorder that has only been described in 37 affected patients. In this report, we describe the presence of an in-frame homozygous variant (c.471_477delTTTAAAAinsG) in the MOCS2 gene in an affected child, diagnosed with Ohtahara syndrome according to the clinical manifestations. The analysis of the three-dimensional structure of the protein and the amino acid substitutions suggested the pathogenicity of this mutation. To prevent transmitting this mutation to the next generation, we used preimplantation genetic testing for the monogenic disorders (PGT-M) protocol to select MOCS2 gene mutant-free embryos for transfer in an in vitro fertilization (IVF) program. As a result, a healthy child was born. Interestingly, both parents of the proband shared an identical mitochondrial (mt) DNA control region, assuming their close relationship and thus suggesting that both copies of the nuclear rare variant c.471_477delTTTAAAAinsG may have been transmitted from the same female ancestor. Our estimation of the a priori probability of meeting individuals with the same mtDNA haplotype confirms the assumption of a possible distant maternal relationship among the proband’s direct relatives.

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.

The Nematoda phylum is a promising group for studying the relationship between ecological and genetic characteristics, but the number of high-quality sequences, including mtDNA, is small. In this work, the possibility of increasing the database for these aims by using barcoding data (COX1) was considered, possible problems and their significance for mtDNA mutagenesis analyses were identified.