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.

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.

Quantitative detection of low-abundance somatic structural variants in normal cells by high-throughput sequencing.

The detection and quantification of low-abundance somatic DNA mutations by high-throughput sequencing is challenging because of the difficulty of distinguishing errors from true mutations. There are several approaches available for analyzing somatic point mutations and small insertions or deletions, but an accurate genome-wide assessment of somatic structural variants (somSVs) in bulk DNA is still not possible. Here we present Structural Variant Search (SVS), a method to accurately detect rare somSVs by low-coverage sequencing. We demonstrate direct quantitative assessment of elevated somSV frequencies induced by known clastogenic compounds in human primary cells.

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.

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.

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.

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.

Expression of retrotransposons contributes to aging in Drosophila.

Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. Here, we use a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, our data show a clear link between retrotransposon expression and aging.

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.

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.

Sept5 deficiency exerts pleiotropic influence on affective behaviors and cognitive functions in mice.

Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression.

Over-expression of a human chromosome 22q11.2 segment including TXNRD2, COMT and ARVCF developmentally affects incentive learning and working memory in mice.

Duplication of human chromosome 22q11.2 is associated with elevated rates of mental retardation, autism and many other behavioral phenotypes. However, because duplications cover 1.5-6 Mb, the precise manner in which segments of 22q11.2 causally affect behavior is not known in humans. We have now determined the developmental impact of over-expression of an approximately 190 kb segment of human 22q11.2, which includes the genes TXNRD2, COMT and ARVCF, on behaviors in bacterial artificial chromosome (BAC) transgenic (TG) mice. BAC TG mice and wild-type (WT) mice were tested for their cognitive capacities, affect- and stress-related behaviors and motor activity at 1 and 2 months of age. An enzymatic assay determined the impact of BAC over-expression on the activity level of COMT. BAC TG mice approached a rewarded goal faster (i.e. incentive learning), but were impaired in delayed rewarded alternation during development. In contrast, BAC TG and WT mice were indistinguishable in rewarded alternation without delays, spontaneous alternation, prepulse inhibition, social interaction, anxiety-, stress- and fear-related behaviors and motor activity. Compared with WT mice, BAC TG mice had an approximately 2-fold higher level of COMT activity in the prefrontal cortex, striatum and hippocampus. These data suggest that over-expression of this 22q11.2 segment enhances incentive learning and impairs the prolonged maintenance of working memory, but has no apparent effect on working memory per se, affect- and stress-related behaviors or motor capacity. High copy numbers of this 22q11.2 segment might contribute to a highly selective set of phenotypes in learning and cognition during development.

A workflow for simultaneous DNA copy number and methylome analysis of inner cell mass and trophectoderm cells from human blastocysts.

OBJECTIVE: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development. DESIGN: Experimental. SETTING: Academic medical center. PATIENT(S): Donated genetically abnormal blastocysts. INTERVENTION(S): Single cells were isolated, followed by bisulfite conversion and sequencing to identify CNV and methylome profiles. MAIN OUTCOME MEASURE(S): CNV and methylation profiling. RESULT(S): Two embryos were dissociated, isolating 46 single cells, with 17 ICM and 12 TE cells selected for further downstream analysis. Chromosome ploidies and embryo sex were concordant with the results from conventional aneuploidy testing. In 3 of the 29 cells, additional aneuploidies were discovered, indicating possible mosaicism undetected by routine preimplantation genetic testing for aneuploidy. CpG methylation frequency was higher in ICM cells compared with TE cells (44.3% vs. 32.4%), respectively, while non-CpG methylation frequency was similar among both cell types. CpG methylation levels accurately distinguished ICM from TE cells epigenetically. CONCLUSION(S): We describe an effective workflow for isolating and sequencing single ICM and TE cells from human blastocysts. The use of methylation profiling can help distinguish these two cell populations better then morphologic identification alone. TE cells had significantly lower levels of DNA methylation, which may be explained in part by the fact that these cells have begun the process of differentiation and are transcriptionally more active than ICM. This approach may be used to explore the genetic complexities within human embryos, specifically among the two primary cell types seen at this stage of development.

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.

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.

A direct comparison of interphase FISH versus low-coverage single cell sequencing to detect aneuploidy reveals respective strengths and weaknesses.

Aneuploidy has been reported to occur at remarkably high levels in normal somatic tissues using Fluorescence In Situ Hybridization (FISH). Recently, these reports were contradicted by single-cell low-coverage whole genome sequencing (scL-WGS) analyses, which showed aneuploidy frequencies at least an order of magnitude lower. To explain these seemingly contradictory findings, we used both techniques to analyze artificially generated mock aneuploid cells and cells with natural random aneuploidy. Our data indicate that while FISH tended to over-report aneuploidies, a modified 2-probe approach can accurately detect low levels of aneuploidy. Further, scL-WGS tends to underestimate aneuploidy levels, especially in a polyploid background.

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.

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung.

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

DARPP-32 phosphorylation opposes the behavioral effects of nicotine.

BACKGROUND: The addictive properties of nicotine are mediated via dopaminergic pathways and their post-synaptic neurons in the striatum. Because post-synaptic neurons within the striatum contain high levels of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), we hypothesized that DARPP-32 may functionally contribute to the behavioral effects of nicotine. METHODS: We examined the behavioral effects of nicotine and the phosphorylation state of DARPP-32 in wild-type (WT) and DARPP-32 knockout (KO) mice. In one experiment, we assessed voluntary nicotine intake (0-50 microg/ml) of WT and KO mice in a two-bottle choice paradigm. In a separate experiment, the motor-depressant effects of acute and repeated nicotine injections (0-.8 mg/kg, subcutaneously [SC]) were assessed. The phosphorylation of DARPP-32 at threonine34 and threonine75 were examined using Western blotting. RESULTS: A heightened responsiveness to nicotine was seen in KO mice when compared with WT mice in oral intake and motor depression. The enhanced responsiveness in KO mice was not due to alterations in taste sensations, fluid intake, or blood nicotine or cotinine levels. Systemic injections of nicotine resulted in increased striatal DARPP-32 phosphorylation at threonine34 and threonine75. CONCLUSIONS: DARPP-32 opposes the behavioral effects of nicotine possibly via concurrent phosphorylation at the two threonine sites.