Age-related Mutation Accumulation Research Articles

Abstract 440: Single-cell whole-genome sequencing reveals somatic mutation signatures in normal somatic cells predictive of cancer later in life

Abstract Single-cell sequencing for analyzing DNA mutations across the genome in somatic tissues is critically important for studying development, cancer and aging. However, current procedures are prone to artifacts and to date a reliable protocol for single-cell somatic mutation analysis remains to be developed. We address the two largest sources of artifacts, i.e., DNA denaturation-related cytosine deamination and allelic bias-driven whole genome amplification errors. We first reconfigured multiple displacement amplification (MDA) into an efficient protocol for whole genome amplification of single cells without cytosine deamination artifacts, i.e., Single Cell MDA (SCMDA). We then developed a new single-cell SNV caller (SCcaller) that distinguishes real somatic mutations and amplification errors by utilizing a SNP-based localized estimate of allelic amplification bias. The procedure was validated by comparing SCMDA-amplified single cells with unamplified clones derived from single cells from the same population. Using this highly accurate single-cell whole-genome sequencing method we analyzed human B lymphocytes from donors varying in age from birth to over 100 year, studying both genome distribution and functional impact of base substitution mutations. Mutations per cell were found to increase with age from less than 500 in cord blood to over 3,000 in cells from individuals over 100. While overall mutations were randomly distributed across the genome with all chromosomes equally affected, 24 hotspot regions were identified, five of which were part of immunoglobulin variable regions subject to somatic hypermutation. Age-related mutation accumulation was found to be significantly slower in genomic sequences directly involved in cellular function, such as exons and gene regulatory sequences. Still, on average, B lymphocytes from aged individuals contained 3-15 damaging mutations in the transcribed part of the exome identified by RNA-seq, plus 15-50 mutations in transcription factor binding sites identified by ATAC-seq. Analysis of the spontaneous mutation spectra in these normal cells revealed signatures similar to those previously found associated with B cell leukemia and other cancers. These results indicate that cancer risk is already part of age-related mutation accumulation in normal cells. Taken together, our single-cell sequencing method provides a firm foundation for analyzing cellular genetic heterogeneity in normal human tissues. Citation Format: Xiao Dong, Lei Zhang, Moonsook Lee, Alexander Y. Maslov, Jan Vijg. Single-cell whole-genome sequencing reveals somatic mutation signatures in normal somatic cells predictive of cancer later in life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 440.

Compromised Tissue Renewal in the Ageing Human Colonic Epithelium

BACKGROUND: The intestinal epithelium is the most rapidly renewing tissue in the body. It is widely believed that this attribute minimises the accumulation of age-related molecular damage. Increasing evidence suggests that this protective mechanism is undermined by agerelated molecular changes that accumulate in long-lived stem/progenitor cells. Age-related molecular damage in the intestinal epithelium of flies and rodents manifests as a hyperproliferative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reduced regenerative potential following damage. Furthermore, the human colonic epithelium is subject to age-related accumulation of mutations in mitochondrial DNA. Given that ageing is a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageing human colonic epithelium has received little attention. AIM: To investigate age-related changes in the renewal of the human colonic epithelium.METHODS: Tissue biopsies obtained at sigmoidoscopy (Ethical approval) from young ( 70 years, N= 12) individuals with no apparent pathology were immediately fixed or processed for crypt isolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy. Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morphometric analysis and immunofluorescence for detection of Ki67 (proliferation marker), beta catenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cell marker). RESULTS: Crypts from young subjects (n=116 crypts) were significantly longer (p< 0.05) than those derived from the older cohort (117 crypts), 375± 10 um versus 330 ± 10 um, respectively. The percentage of Ki67 positive cells in all regions along the cryptaxis was significantly greater (p<0.05) in tissue from older subjects : e.g. crypt base 35% (young) versus 52% (old); mid region 42%% (young) versus 62% (old) and upper region 10% (young) versus 17% (old). The number of cell divisions observed under timelapse microscopy was reduced for crypts derived from older subjects suggesting that the increased Ki67 labelling index in the older group reflected a slower cell cycle time. Although the nature of the proliferating cell type is not known at this stage, an increase in OLFM4+ (stem) cell number was observed along the axis of crypts derived from older subjects. The above traits were associated with a trend towards an extended profile of immunolabelling intensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis of tissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length, cell proliferation and markers for intestinal stem cells and Wnt signalling components along the crypt-axis suggest that tissue renewal is compromised in the ageing human colonic epithelium.

Age-Related Accumulation of Mutations Supports a Replication-Dependent Mechanism of Spontaneous Mutation at Tandem Repeat DNA Loci in Mice

Expanded simple tandem repeat (ESTR) loci belong to the class of highly unstable loci in the mouse genome. The mechanisms underlying the very high spontaneous instability at these loci still remain poorly understood. Using single-molecule polymerase chain reaction, here we have compared the pattern of mutation accumulation in tissues with different proliferation capacities in male mice of age 12, 26, 48, and 96 weeks. In the nonproliferating brain, we did not observe any measurable age-related accumulation of ESTR mutations. In contrast, a highly elevated frequency of ESTR mutation was detected in the sperm samples taken from old mice; similar changes were also observed in the bone marrow tissue. The spectra of ESTR mutations accumulated in all tissues of young and old mice did not significantly differ. Taken together, these data clearly imply that spontaneous ESTR mutations occur almost exclusively in replication-proficient cells. To gain further insights into the mechanisms of ESTR mutation, we developed a stochastic model of age-related mutation accumulation. The observed spectra of ESTR mutants accumulated in the brain and sperm were fairly accurately approximated assuming the values of ESTR mutation rate, ranging from 0.01 to 0.04 per cell division. As these estimates dramatically exceed those for protein-coding genes and microsatellite loci, our data therefore suggest that ESTRs represent one of the most unstable loci in the mammalian genome. The results of our study also imply that ESTR loci can be regarded as a class of expanded microsatellites, with the mechanism of spontaneous mutation most probably attributed to replication slippage.

XPC is involved in genome maintenance through multiple pathways in different tissues

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.

Intra-Organ Variation in Age-Related Mutation Accumulation in the Mouse

Using a transgenic mouse model harboring chromosomally integrated lacZ mutational target genes, we previously demonstrated that mutations accumulate with age much more rapidly in the small intestine than in the brain. Here it is shown that in the small intestine point mutations preferentially accumulate in epithelial cells of the mucosa scraped off the underlying serosa. The mucosal cells are the differentiated villus cells that have undergone multiple cell divisions. A smaller age-related increase, also involving genome rearrangements, was observed in the serosa, which consists mainly of the remaining crypts and non-dividing smooth muscle cells. In the brain we observed an accumulation of only point mutations in no other areas than hypothalamus and hippocampus. To directly test for cell division as the determining factor in the generation of point mutations we compared mutation induction between mitotically active and quiescent embryonic fibroblasts from the same lacZ mice, treated with either UV (a point mutagen) or hydrogen peroxide (a clastogen). The results indicate that while point mutations are highly replication-dependent, genome rearrangements are as easily induced in non-dividing cells as in mitotically active ones. This strongly suggests that the point mutations found to have accumulated in the mucosal part of the small intestine are the consequence of replication errors. The same is likely true for point mutations accumulating in hippocampus and hypothalamus of the brain since neurogenesis in these two areas continues throughout life. The observed intra-organ variation in mutation susceptibility as well as the variation in replication dependency of different types of mutations indicates the need to not only extend observations made on whole organs to their sub-structures but also take the type of mutations and mitotic activity of the cells into consideration. This should help elucidating the impact of genome instability and its consequences on aging and disease.

Age related microsatellite instability in T cells from healthy individuals

Many immune functions decline with age and may jeopardize the elderly, as illustrated, for example by the significantly higher mortality rate from influenza in old age. Although innate and humoral immunity are affected by aging, it is the T cell compartment, which manifests most alterations. The mechanisms behind these alterations are still unclear, and several explanations have been offered including thymic involution and Telomere attrition leading to cell senescence. Age related accumulation of mutations has been documented and could serve as an additional mechanism of T cell dysfunction. One effective repair mechanism capable of rectifying errors in DNA replications is the mismatch repair (MMR) system. We previously reported a comparative examination of individual DNA samples from blood cells obtained at 10 year intervals from young and old subjects. We showed significantly higher rates of microsatellite instability (MSI), an indicator of MMR dysfunction in older subjects, compared to young. In the present study we confirm this result, using direct automated sequencing and in addition, we demonstrate that as CD8 lymphocytes from aged individuals, undergo repeated population doublings (PDs) in culture, they develop MSI. CD4 clones that also undergo repeated PDs in culture develop significant MSI as well. Elucidation of this previously unexplored facet of lymphocyte dynamics in relation to aging may help identify novel mechanisms of immunosenescence and pathways that could serve as targets for interventions to restore immune function.

Age-related mutation accumulation at a lacZ reporter locus in normal and tumor tissues of Trp53-deficient mice

Increased genomic instability has been found associated with cancer and aging. The p53 tumor suppressor protein is a major determinant of genomic instability as a regulator of cell cycle control and apoptosis in response to DNA damage. To investigate the rate of age-related mutation accumulation in the absence of p53, we crossed Trp53 null mice with transgenic mice harboring a lacZ mutational target gene. In the hybrid animals, lacZ mutation frequencies at early age (i.e. at about 2 months) were found to be the same as in the control lacZ animals. However, up until about 6 months, when the Trp53-knockout mice usually die from cancer, mutations were found to accumulate with age in the spleen, and to a lesser extent in the liver, at a more rapid rate than in the control Trp53 +/+ or Trp53 +/−, lacZ hybrid mice. Treatment of 2–3-month-old Trp53 −/−, lacZ hybrid mice with the powerful mutagen ethyl nitrosourea (ENU) resulted in a higher number of mutations induced in the liver but not in the spleen, as compared to the Trp53 +/+, lacZ mice. These results suggest that p53 is not an important determinant of gene mutation induction, either spontaneously during development or after treatment with a mutagen. The accelerated age-related accumulation of mutations in normal spleen and liver could be explained by the defect in apoptosis, which would prevent severely damaged cells from being eliminated.

Mutation frequency and type during ageing in mouse seminiferous tubules

Mutations arise in the germline by errors of replication, recombination and repair, and the movement of transposable elements. Transgenic mice bearing reporter genes such as lacZ have proven useful for measurements of spontaneous and induced mutation frequencies, as well as studies of the effects of ageing. In this study, testicular DNA from lacZ transgenic mice was examined for age-related effects on mutation frequency and type. The recovered transgene was tested for simple substitutions and rearrangements including transposition of endogenous mobile elements. There was no evidence for either an age-related accumulation of mutations, or for the insertion of retrotransposons into the lacZ reporter gene in the testis. We conclude that the frequency of retrotransposition of several mouse mobile elements into the lacZ reporter gene is less than 3.73×10 −8. This is significantly less than the known frequency of ∼7% of all spontaneous mutations in the mouse being due to retrotransposition of these elements.

Ageing and the mismatch repair system

Age-related accumulation of mutations has been extensively documented, and it has been proposed as one of the prominent causes of malignancies in old age. The present review is focused on the particular case of DNA mismatch repair system (MMR), that has drawn increased attention for its possible relevance to malignancy. We also report on our own observations on an age-associated genomic instability that develops with age in the MMR system. Our study was performed on DNA samples that were prepared from peripheral blood cells, obtained at a 10-year interval from young and old human subjects. The two DNA samples from each individual were examined comparatively. The older individuals showed a significantly higher rate of microsatellite instability (MSI) in several loci examined, whereas no difference was found between the paired samples of any of the young subjects. We suggest that this increase in MSI with age may indicate an overall genomic instability in the elderly.

Estimation of mutation frequencies in normal mammalian cells and the development of cancer

A current controversy in cancer research is whether the rate of accumulation of mutations in normal somatic cells is sufficient to account for the number of mutations in malignant cells. This review will focus on the types of mutations that can occur in mammalian somatic cells and the frequency at which some of these mutations have been shown to occur in vivo. Human and mouse mutation detection systems will be highlighted. The possibility that distinct mutational events can arise from a common precursor will be discussed, as will the possibility that inherited and sporadic cancers can acquire mutator phenotypes at different times in tumor development. Consideration will also be given to a potential relationship between an age-related accumulation of mutations and cancer.

Transgenic mouse models for studying mutations in vivo: applications in aging research

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recovery the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Age-related accumulation of mutations in human T-lymphocytes.

Annals of the New York Academy of SciencesVolume 663, Issue 1 p. 36-42 Age-Related Accumulation of Mutations in Human T-Lymphocytesa H. VRIELING, H. VRIELING MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the Netherlands J. A. Cohen Institute Interuniversity Research Institute for Radiopathology and Radiation Protection Leiden, the NetherlandsSearch for more papers by this authorA. D. TATES, A. D. TATES MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the NetherlandsSearch for more papers by this authorA. T. NATARAJAN, A. T. NATARAJAN MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the Netherlands J. A. Cohen Institute Interuniversity Research Institute for Radiopathology and Radiation Protection Leiden, the NetherlandsSearch for more papers by this authorA. A. VAN ZEELAND, A. A. VAN ZEELAND MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the Netherlands J. A. Cohen Institute Interuniversity Research Institute for Radiopathology and Radiation Protection Leiden, the NetherlandsSearch for more papers by this author H. VRIELING, H. VRIELING MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the Netherlands J. A. Cohen Institute Interuniversity Research Institute for Radiopathology and Radiation Protection Leiden, the NetherlandsSearch for more papers by this authorA. D. TATES, A. D. TATES MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the NetherlandsSearch for more papers by this authorA. T. NATARAJAN, A. T. NATARAJAN MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the Netherlands J. A. Cohen Institute Interuniversity Research Institute for Radiopathology and Radiation Protection Leiden, the NetherlandsSearch for more papers by this authorA. A. VAN ZEELAND, A. A. VAN ZEELAND MGC-Department of Radiation Genetics and Chemical Mutagenesis Wassenaarseweg 72, 2333 AL Leiden, State University of Leiden Leiden, the Netherlands J. A. Cohen Institute Interuniversity Research Institute for Radiopathology and Radiation Protection Leiden, the NetherlandsSearch for more papers by this author First published: November 1992 https://doi.org/10.1111/j.1749-6632.1992.tb38646.xCitations: 5 a This work was supported by grants from the Dutch Cancer Society, the Environmental Research Programme of the European Community, Shell International B.V., and the J. A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden, the Netherlands. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume663, Issue1Aging and Cellular Defense MechanismsNovember 1992Pages 36-42 RelatedInformation