Negative Selection Allows DNA Mismatch Repair-Deficient Mouse Fibroblasts In Vitro to Tolerate High Levels of Somatic Mutations.

Substantial numbers of somatic mutations have been found to accumulate with age in different human tissues. Clonal cellular amplification of some of these mutations can cause cancer and other diseases. However, it is as yet unclear if and to what extent an increased burden of random mutations can affect cellular function without clonal amplification. We tested this in cell culture, which avoids the limitation that an increased mutation burden in vivo typically leads to cancer. We performed single-cell whole-genome sequencing of primary fibroblasts from DNA mismatch repair (MMR) deficient Msh2-/- mice and littermate control animals after long-term passaging. Apart from analyzing somatic mutation burden we analyzed clonality, mutational signatures, and hotspots in the genome, characterizing the complete landscape of somatic mutagenesis in normal and MMR-deficient mouse primary fibroblasts during passaging. While growth rate of Msh2-/- fibroblasts was not significantly different from the controls, the number of de novo single-nucleotide variants (SNVs) increased linearly up until at least 30,000 SNVs per cell, with the frequency of small insertions and deletions (INDELs) plateauing in the Msh2-/- fibroblasts to about 10,000 INDELS per cell. We provide evidence for negative selection and large-scale mutation-driven population changes, including significant clonal expansion of preexisting mutations and widespread cell-strain-specific hotspots. Overall, our results provide evidence that increased somatic mutation burden drives significant cell evolutionary changes in a dynamic cell culture system without significant effects on growth. Since similar selection processes against mutations preventing organ and tissue dysfunction during aging are difficult to envision, these results suggest that increased somatic mutation burden can play a causal role in aging and diseases other than cancer.

Evidence for negative selection in human primary fibroblasts to tolerate high somatic mutation loads upon treatment with multiple low doses of N-ethyl-N-nitrosourea.

High-throughput sequencing at the single-cell and single-molecule level has shown that mutation rate is much higher in somatic cells than in the germline, with thousands of mutations accumulating with age in most human tissues. While there is now ample evidence that some of these mutations can clonally amplify and lead to disease, most notably cancer, the total burden of mutations a cell can tolerate without functional decline remains unknown. Here we addressed this question by exposing human primary fibroblasts multiple times to low doses of N-ethyl-N-nitrosourea (ENU) and quantitatively analyzing somatic mutation burden using single-cell whole genome sequencing. The results indicate that individual cells can sustain ∼60,000 single-nucleotide variants (SNVs) with only a slight adverse effect on growth rate. We found evidence for selection against potentially deleterious variants in gene coding regions as well as depletion of mutations in sequences associated with genetic pathways expressed in these human fibroblasts, most notably those relevant for maintaining basic cellular function and growth. However, no evidence of negative selection was found for variants in non-coding regions. We conclude that actively proliferating fibroblasts can tolerate very high levels of somatic mutations without major adverse effects on growth rate via negative selection against damaging coding mutations. Since most tissues in adult organisms have very limited capacity to select against mutations based on a growth disadvantage, these results suggest that a causal effect of somatic mutations in aging and disease cannot be ruled out.

Analyzing somatic mutations by single-cell whole-genome sequencing.

Somatic mutations are the cause of cancer and have been implicated in other, noncancerous diseases and aging. While clonally expanded mutations can be studied by deep sequencing of bulk DNA, very few somatic mutations expand clonally, and most are unique to each cell. We describe a detailed protocol for single-cell whole-genome sequencing to discover and analyze somatic mutations in tissues and organs. The protocol comprises single-cell multiple displacement amplification (SCMDA), which ensures efficiency and high fidelity in amplification, and the SCcaller software tool to call single-nucleotide variations and small insertions and deletions from the sequencing data by filtering out amplification artifacts. With SCMDA and SCcaller at its core, this protocol describes a complete procedure for the comprehensive analysis of somatic mutations in a single cell, covering (1) single-cell or nucleus isolation, (2) single-cell or nucleus whole-genome amplification, (3) library preparation and sequencing, and (4) computational analyses, including alignment, variant calling, and mutation burden estimation. Methods are also provided for mutation annotation, hotspot discovery and signature analysis. The protocol takes 12-15 h from single-cell isolation to library preparation and 3-7 d of data processing. Compared with other single-cell amplification methods or single-molecular sequencing, it provides high genomic coverage, high accuracy in single-nucleotide variation and small insertions and deletion calling from the same single-cell genome, and fewer processing steps. SCMDA and SCcaller require basic experience in molecular biology and bioinformatics. The protocol can be utilized for studying mutagenesis and genome mosaicism in normal and diseased human and animal tissues under various conditions.

Somatic mutation burden in relation to aging and functional life span: implications for cellular reprogramming and rejuvenation.

The accrual of somatic mutations has been implicated as causal factors in aging since the 1950s. However, the quantitative analysis of somatic mutations has posed a major challenge due to the random nature of de novo mutations in normal tissues, which has limited analysis to tumors and other clonal lineages. Advances in single-cell and single-molecule next-generation sequencing now allow to obtain, for the first time, detailed insights into the landscape of somatic mutations in different human tissues and cell types as a function of age under various conditions. Here, we will briefly recapitulate progress in somatic mutation analysis and discuss the possible relationship between somatic mutation burden with functional life span, with a focus on differences between germ cells, stem cells, and differentiated cells.

Concurrent analysis of genome and transcriptome in one single cell.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps - 1) immobilization of a single cell on solid substrate, 2) hypotonic lysis of immobilized single cell, and 3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.

Mitigating age-related somatic mutation burden.

Genomes are inherently unstable and require constant DNA repair to maintain their genetic information. However, selective pressure has optimized repair mechanisms in somatic cells only to allow transmitting genetic information to the next generation, not to maximize sequence integrity long beyond the reproductive age. Recent studies have confirmed that somatic mutations, due to errors during genome repair and replication, accumulate in tissues and organs of humans and model organisms. Here, we describe recent advances in the quantitative analysis of somatic mutations in vivo. We also review evidence for or against a possible causal role of somatic mutations in aging. Finally, we discuss options to prevent, delay or eliminate de novo, random somatic mutations as a cause of aging.

Female reproductive tract microbiome and early miscarriages.

Miscarriage is one of the main causes of reproductive loss, which can lead to a number of physical and psychological complications and other long-term consequences. However, the role of vaginal and uterine microbiome in such complications is poorly understood. To review the published data on the function of the female reproductive tract microbiome in the pathogenesis of early miscarriages. The articles published over the past 20 years and deposited in PubMed, Google Academy, Scopus, Elibrary, ResearchGate, and EBSCO databases were analyzed. The review presents new data on the impact of the vaginal and uterine microbiome on the local immunity, including defense against sexually transmitted infections, and its association with other factors of miscarriages. The studies on the microbiome of non-pregnant women with recurrent miscarriages in the anamnesis, patients undergoing IVF, and pregnant women with miscarriages, as well as new directions in the microbiome research are discussed. The majority of studies have demonstrated that the dominant species of the vaginal and uterine microbiome in patients with early miscarriages are non-Lactobacillus bacteria. As many of these bacteria have not previously been detected by cultural studies and their role in obstetric complications is not well defined, further research on the female reproductive tract microbiome, including the microbiome of the cervix uteri, is needed to develop new approaches for the prognosis and prevention of miscarriages.

Transcriptional profiling reveals ataxia telangiectasia mutated pathways regulate joint copper and arsenic toxicity for hepatic metalloplasia and anti-cancer therapies.

AIMS: Exposures to toxic metals, including arsenic (As), pose health risks but joint effects of physiologically needed metals, e.g., copper (Cu), are ill-defined for regulated metal-dependent cell proliferation (or metalloplasia). This study elucidated hepatic toxicities of As and Cu. MAIN METHODS: Human HuH-7 cells were exposed to As and Cu and mRNA profiling obtained for molecular networks, regulators and signaling pathways. This followed biological testing of ATM signaling-related DNA damage response, mitochondrial dysfunction and lysosome activity using HuH-7 cells and primary hepatocytes. Free Cu ions were bound to 3-indole propionic acid for finding their contribution in toxicity. KEY FINDINGS: The As or As plus Cu toxicities in HuH-7 cells produced dimorphic down- or up-regulation patterns in mRNA profiles. Significant differences extended for ontologies in protein synthesis, intermediary metabolism, mitochondrial function, autophagy, or cell survival and growth. Bioassays revealed ATM signaling regulated As and Cu toxicity for oxidative phosphorylation, mitochondrial membrane potential, lysosomal activity, DNA damage response, and cell growth-arrest. Removal of reactive Cu ions decreased As and Cu toxicity. Primary hepatocytes withstood Cu and As toxicity better. SIGNIFICANCE: This joint As and Cu toxicity offers further mechanisms for metalloplasia, carcinogenesis and tissue damage in other settings, e.g., during excess Cu accumulation in Wilson disease. Moreover, joint As and Cu toxicities are relevant for anti-cancer therapies, potentially including manipulations to increase intracellular Cu through altered uptake or efflux processes and incorporating ATM-related checkpoint inhibitors. Superior tolerance of healthy hepatocytes to Cu and As toxicity should improve safety margins for anti-cancer therapies.

Single-cell analysis of somatic mutations in human bronchial epithelial cells in relation to aging and smoking.

Although lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here, we applied single-cell whole-genome sequencing of proximal bronchial basal cells from 33 participants aged between 11 and 86 years with smoking histories varying from never-smoking to 116 pack-years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers. When plotted against smoking pack-years, mutations followed the linear increase in cancer risk until about 23 pack-years, after which no further increase in mutation frequency was observed, pointing toward individual selection for mutation avoidance. Known lung cancer-defined mutation signatures tracked with both age and smoking. No significant enrichment for somatic mutations in lung cancer driver genes was observed.

Single-molecule, quantitative detection of low-abundance somatic mutations by high-throughput sequencing.

Postzygotic somatic mutations have been found associated with human disease, including diseases other than cancer. Most information on somatic mutations has come from studying clonally amplified mutant cells, based on a growth advantage or genetic drift. However, almost all somatic mutations are unique for each cell, and the quantitative analysis of these low-abundance mutations in normal tissues remains a major challenge in biology. Here, we introduce single-molecule mutation sequencing (SMM-seq), a novel approach for quantitative identification of point mutations in normal cells and tissues.

Single-cell analysis of somatic mutation burden in mammary epithelial cells of pathogenic BRCA1/2 mutation carriers.

Inherited germline mutations in the breast cancer gene 1 (BRCA1) or BRCA2 genes (herein BRCA1/2) greatly increase the risk of breast and ovarian cancer, presumably by elevating somatic mutational errors as a consequence of deficient DNA repair. However, this has never been directly demonstrated by a comprehensive analysis of the somatic mutational landscape of primary, noncancer, mammary epithelial cells of women diagnosed with pathogenic BRCA1/2 germline mutations. Here, we used an accurate, single-cell whole-genome sequencing approach to first show that telomerized primary mammary epithelial cells heterozygous for a highly penetrant BRCA1 variant displayed a robustly elevated mutation frequency as compared with their isogenic control cells. We then demonstrated a small but statistically significant increase in mutation frequency in mammary epithelial cells isolated from the breast of BRCA1/2 mutation carriers as compared with those obtained from age-matched controls with no genetically increased risk for breast cancer.

SomaMutDB: a database of somatic mutations in normal human tissues.

De novo mutations, a consequence of errors in DNA repair or replication, have been reported to accumulate with age in normal tissues of humans and model organisms. This accumulation during development and aging has been implicated as a causal factor in aging and age-related pathology, including but not limited to cancer. Due to their generally very low abundance mutations have been difficult to detect in normal tissues. Only with recent advances in DNA sequencing of single-cells, clonal lineages or ultra-high-depth sequencing of small tissue biopsies, somatic mutation frequencies and spectra have been unveiled in several tissue types. The rapid accumulation of such data prompted us to develop a platform called SomaMutDB (https://vijglab.einsteinmed.org/SomaMutDB) to catalog the 2.42 million single nucleotide variations (SNVs) and 0.12 million small insertions and deletions (INDELs) thus far identified using these advanced methods in nineteen human tissues or cell types as a function of age or environmental stress conditions. SomaMutDB employs a user-friendly interface to display and query somatic mutations with their functional annotations. Moreover, the database provides six powerful tools for analyzing mutational signatures associated with the data. We believe such an integrated resource will prove valuable for understanding somatic mutations and their possible role in human aging and age-related diseases.

Maintenance of genome sequence integrity in long- and short-lived rodent species.

DNA mutations in somatic cells have been implicated in the causation of aging, with longer-lived species having a higher capacity to maintain genome sequence integrity than shorter-lived species. In an attempt to directly test this hypothesis, we used single-cell whole-genome sequencing to analyze spontaneous and bleomycin-induced somatic mutations in lung fibroblasts of four rodent species with distinct maximum life spans, including mouse, guinea pig, blind mole-rat, and naked mole-rat, as well as humans. As predicted, the mutagen-induced mutation frequencies inversely correlated with species-specific maximum life span, with the greatest difference observed between the mouse and all other species. These results suggest that long-lived species are capable of processing DNA damage in a more accurate way than short-lived species.

Age-related telomere attrition causes aberrant gene expression in sub-telomeric regions.

Telomere attrition has been proposed as a biomarker and causal factor in aging. In addition to causing cellular senescence and apoptosis, telomere shortening has been found to affect gene expression in subtelomeric regions. Here, we analyzed the distribution of age-related differentially expressed genes from the GTEx RNA sequencing database of 54 tissue types from 979 human subjects and found significantly more upregulated than downregulated genes in subtelomeric regions as compared to the genome-wide average. Our data demonstrate spatial relationships between telomeres and gene expression in aging.

FOXO3a acts to suppress DNA double-strand break-induced mutations.

Genomic instability is one of the hallmarks of aging, and both DNA damage and mutations have been found to accumulate with age in different species. Certain gene families, such as sirtuins and the FoxO family of transcription factors, have been shown to play a role in lifespan extension. However, the mechanism(s) underlying the increased longevity associated with these genes remains largely unknown and may involve the regulation of responses to cellular stressors, such as DNA damage. Here, we report that FOXO3a reduces genomic instability in cultured mouse embryonic fibroblasts (MEFs) treated with agents that induce DNA double-strand breaks (DSBs), that is, clastogens. We show that DSB treatment of both primary human and mouse fibroblasts upregulates FOXO3a expression. FOXO3a ablation in MEFs harboring the mutational reporter gene lacZ resulted in an increase in genome rearrangements after bleomycin treatment; conversely, overexpression of human FOXO3a was found to suppress mutation accumulation in response to bleomycin. We also show that overexpression of FOXO3a in human primary fibroblasts decreases DSB-induced γH2AX foci. Knocking out FOXO3a in mES cells increased the frequency of homologous recombination and non-homologous end-joining events. These results provide the first direct evidence that FOXO3a plays a role in suppressing genome instability, possibly by suppressing genome rearrangements.

Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan.

Accumulation of mutations in somatic cells has been implicated as a cause of aging since the 1950s. However, attempts to establish a causal relationship between somatic mutations and aging have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan using a highly accurate single-cell whole-genome sequencing method. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, were located at Ig genes associated with somatic hypermutation (SHM). B cell-specific mutation signatures associated with development, aging, or SHM were found. The SHM signature strongly correlated with the signature found in human B cell tumors, indicating that potential cancer-causing events are already present even in B cells of healthy individuals. We also identified multiple mutations in sequence features relevant to cellular function (i.e., transcribed genes and gene regulatory regions). Such mutations increased significantly during aging, but only at approximately one-half the rate of the genome average, indicating selection against mutations that impact B cell function. This full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

Genes and Pathways Promoting Long-Term Liver Repopulation by Ex Vivo hYAP-ERT2 Transduced Hepatocytes and Treatment of Jaundice in Gunn Rats.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV- (dipeptidyl peptidase-4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)-deficient Gunn rats (model of Crigler-Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti-hYAP-ERT2 (mutated estrogen receptor ligand-binding domain 2)-transduced hepatocytes (YAP-Hc). Yap (yes-associated protein) nuclear translocation/function in YAP-Hc was regulated by tamoxifen. Repopulating YAP-Hc and host hepatocytes were fluorescence-activated cell sorting-purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP-Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte-specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up-regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP-Hc. In Gunn rats transplanted with YAP-Hc+tamoxifen, there was a 65%-81% decline in serum bilirubin over 6 months versus 8%-20% with control-Hc, representing a 3-4-fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1-positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen-regulated nuclear translocation/function of hYAP-ERT2 enabled long-term repopulation of DPPIV-/- and Gunn rat livers by hYAP-ERT2-transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.

Targeted sequencing to discover germline variants in the BRCA1 and BRCA2 genes in a Russian population and their association with breast cancer risk.

BRCA1 and BRCA2 are tumor suppressor genes involved in the repair of DNA damage and transcriptional regulation of the cell cycle. Alterations in BRCA1/2 lead to production of functionally defective proteins that impair DNA repair. Certain mutant variants of BRCA1/2 are strongly associated with increased risk of breast and ovarian cancers, with emerging data on association with other types of cancer. However, variability of BRCA1/2 in Russian populations remains understudied. In this study, we performed targeted sequencing of BRCA1/2 in 145 breast cancer (BC) patients with a family history of BRCA-associated cancers and 47 age-matched cancer-free control individuals with or without a family history of cancer. Subjects for this study were recruited in the Voronezh region of the Russian Federation. We found that two polymorphic variants, rs1799967 (BRCA1) and rs4987117 (BRCA2), were strongly associated with the risk of BC. Both variants have not been previously reported as associated with risk of breast cancer. Presence of the rs4987117 variant increases risk of breast cancer onset (OR = 2.76, p-value = 0.022). Notably, although variant rs80357906 (5382InsC) has been reported as a risk factor for hereditary BC, it was not significantly associated with breast cancer risk in our population (p = 0.192). We also found 12 novel polymorphic variants in BRCA1/2 genes (2 in BRCA1 and 10 in BRCA2). However, none of these variants demonstrated association with the disease. Five germline variants were observed at high frequency (mean AF = 67.14%) and therefore can be considered as a common haplotype in the Voronezh region of the Russian Federation. In summary, our study demonstrates that known pathological variants of BRCA1/2 genes may not be reflective of breast cancer risk assessment when applied to the Russian population. Further, more extended population-specific studies are needed to reveal the reliable list of BRCA1/2 polymorphisms associated with risk of breast cancer in the Russian population.

Bleomycin-induced genome structural variations in normal, non-tumor cells.

Many anticancer drugs are genotoxic agents inducing DNA breaks in actively proliferating cancer cells. However, these same drugs also induce mutations, mostly genome structural variations (GSVs). The detection of GSVs in normal cells and tissues is a major challenge due to the very low abundance of these mutations, which are essentially only detectable in clonal outgrowths, such as tumors. Previously we developed Structural Variant Search (SVS) - an NGS-based assay for the quantitative detection of somatic GSVs in normal cells. Using an improved version of SVS we now demonstrate that the same dose of the anti-cancer drug bleomycin induces about 5 times more somatic GSVs in quiescent primary human fibroblasts than in proliferating cells. GVS induction in non-dividing, normal cells was subsequently confirmed in vivo by demonstrating that a single dose of bleomycin leads to a significant increase of GSV frequency in mouse liver and heart, two postmitotic tissues. Our findings suggest that normal non-cycling differentiated cells may serve as a reservoir of iatrogenically induced mutations. These results provide more insight into the possible molecular mechanisms that underlie late-life morbidities in cancer survivors exposed to chemotherapy.

Accurate identification of single-nucleotide variants in whole-genome-amplified single cells.

Mutation analysis in single-cell genomes is prone to artifacts associated with cell lysis and whole-genome amplification. Here we addressed these issues by developing single-cell multiple displacement amplification (SCMDA) and a general-purpose single-cell-variant caller, SCcaller (https://github.com/biosinodx/SCcaller/). By comparing SCMDA-amplified single cells with unamplified clones from the same population, we validated the procedure as a firm foundation for standardized somatic-mutation analysis in single-cell genomics.