Somatic mutation rates scale with lifespan across mammals

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans. Comparative analyses can shed light on the diversity of mutagenesis across species and on long-standing hypotheses regarding the evolution of somatic mutation rates and their role in cancer and ageing. Here, we used whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found somatic mutagenesis to be dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans, although the relative contribution of each signature varied across species. Remarkably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied displaying a comparable association. Despite widely different life histories among the species surveyed, including ~30-fold variation in lifespan and ~40,000-fold variation in body mass, the somatic mutation burden at the end of lifespan varied only by a factor of ~3. These data unveil common mutational processes across mammals and suggest that somatic mutation rates are evolutionarily constrained and may be a determinant of lifespan.

KARYOTYPAL VARIABILITY OF CARPATHIAN BROWN COWS

Brown Carpathian breed of cattle bred in Transcarpathia at the end of the XIX century. and belongs to the breeds of dairy and meat productivity. Animals of the modern breed are direct descendants of the ancient Brown Carpathian cattle, common at one time throughout Central Europe. The main advantage of the Brown Carpathian breed is that it can be effectively grown on natural pastures – both in the lowlands and in the mountains. The genetics of these animals are resistant to acute infectious diseases, they are well adapted to local conditions, sensitive to improved housing and feeding conditions, and cows produce special milk, which is used in the manufacture of high quality hard cheeses and baby food. Animals of the Brown Carpathian breed belong to the local small domestic breeds and are in a state of significant risk. The uterine population is bred only in households. The aim of our work was to study the karyotype variability of Brown Carpathian cows, which is quite relevant today. Cytogenetic analysis, which was aimed at establishing the karyotypic variability of Brown Carpathian cows, was performed in the village. Nyzhni Vorota, Volovets district, Zakarpattia region, in households. Purebred cows of Brown Carpathian breed – (16 heads) and local animals – (11 heads) were studied. According to the results of cytogenetic analysis, genomic disorders, aneuploidy, were found in purebred cows of Brown Carpathian breed, which was 1.6% and was expressed mainly by hypoploid cells 2n = 56–58. For domestic animals, the frequency of metaphase plates with aneuploidy was 3.3%, which corresponds to a spontaneous level of cytogenetic variability. Structural chromosome abnormalities, chromosomal breaks, in purebred cows were equal to 0.76%, which does not exceed the spontaneous level of chromosomal variability. In local animals, this variability did not manifest itsel. The indicators of the micronucleus test (the proportion of lymphocytes with a micronucleus of 1.7–2.0‰, dinuclear lymphocytes 1.5–2.7‰, and the mitotic index of 4.8–5.5‰, respectively) in domestic animals are higher compared to purebred animals, however, do not exceed those of the species Bos taurus. According to the results of the cytogenetic analysis, it was established that purebred cows of Brown Carpathian breed and their crossbreeds were characterized by quantitative and structural chromosome disorders. Genomic disorders, aneuploidy, in local animals are 2 times higher (3.3%) compared to purebred cows of Brown Carpathian breed with a statistically significant difference in mean values (P > 0.99). Low level or absence of structural disorders of chromosomes in purebred animals and their crossbreeds indicates a low degree of somatic mutagenesis. The indicators of the micronucleus test, as an indicator of the effect of the total mutagenic load on the body of the studied cows, in domestic animals are higher compared to purebred animals, however, do not exceed those characteristic of the species Bos taurus. It was established that the level of somatic mutagenesis is lower and the karyotype is more stable in purebred animals of the Brown Carpathian breed in comparison with the local ones.

Tamoxifen treatment in the neonatal period affects glucose homeostasis in adult mice in a sex-dependent manner

Tamoxifen is a selective estrogen receptor modulator used to activate the CRE ERT2 recombinase, allowing tissue-specific and temporal control of the somatic mutagenesis to generate transgenic mice. Studies integrating development and metabolism require a genetic modification induced by a neonatal tamoxifen administration. Here, we investigate the effects of a neonatal tamoxifen administration on energy homeostasis in adult male and female C57BL/6J mice. C57BL/6J male and female mouse pups received a single injection of tamoxifen one day after birth (NTT) and were fed a high-fat/high-sucrose (HF/HS) diet at 6 weeks of age. We measured weight, body composition, glucose and insulin tolerance, basal metabolism and tibia length and weight in adult mice. The neonatal tamoxifen administration exerted long-term, sex-dependent effects on energy homeostasis. NTT female mice became overweight and developed impaired glucose control in comparison to vehicle-treated littermates. NTT females exhibited 60% increased fat mass, increased food intake, decreased physical activity and decreased energy expenditure, impaired glucose and insulin tolerance, and fasting hyperglycemia and hyperinsulinemia. In contrast, NTT male mice exhibited a modest amelioration of glucose and insulin tolerance, and long-term decreased lean mass linked to decreased bone weight. These results suggest that the neonatal tamoxifen administration exerted a marked and sex-dependent influence on adult energy homeostasis and bone weight, and must therefore be used with caution for the development of transgenic mouse models regarding studies on energy homeostasis and bone biology.

Somatic mutation landscapes at single-molecule resolution

Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Adult zebrafish are widely used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 “crispants”. We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.

Applications of Single-Cell DNA Sequencing

Over the past decade, genomic analyses of single cells—the fundamental units of life—have become possible. Single-cell DNA sequencing has shed light on biological questions that were previously inaccessible across diverse fields of research, including somatic mutagenesis, organismal development, genome function, and microbiology. Single-cell DNA sequencing also promises significant future biomedical and clinical impact, spanning oncology, fertility, and beyond. While single-cell approaches that profile RNA and protein have greatly expanded our understanding of cellular diversity, many fundamental questions in biology and important biomedical applications require analysis of the DNA of single cells. Here, we review the applications and biological questions for which single-cell DNA sequencing is uniquely suited or required. We include a discussion of the fields that will be impacted by single-cell DNA sequencing as the technology continues to advance. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 22 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

ABSTRACTAdult zebrafish are increasingly used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 ‘crispants’. We find that capillary electrophoresis is a reliable method to measure indel frequencies, while avoiding the limitations of restriction enzyme-based genotyping. Using a quantifiable behavioral assay, we identify 7 single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.

A body map of somatic mutagenesis in morphologically normal human tissues

Somatic mutations accumulated in normal tissues are associated with aging and disease. Here, we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies (~ 600 cells each), mostly from the epithelia, of nine organs from five donors. We found that somatic mutation accumulations and clonal expansions are widespread, although with variable extent, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for tissues from esophagus and cardia. Endogenous mutational processes like SBS1 and SBS5 are ubiquitous among normal tissues though exhibiting different relative activities. Exogenous mutational processes like SBS22 were found in different tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimeter resolution. In epithelial tissues from esophagus and cardia, macroscopic somatic clones expanded to several millimeters were frequently seen, whereas in tissues from colon, rectum, and duodenum somatic clones were microscopic in size and evolved independently. Our study depicted a body map of somatic mutations and clonal expansions from the same individuals, and it revealed that the degree of somatic clonal expansion and enrichment of driver mutations are highly organ specific.

Clonal Hematopoiesis, Cardiovascular Diseases and Hematopoietic Stem Cells

Cardiovascular diseases and cancer, the leading causes of morbidity and mortality in the elderly, share some common mechanisms, in particular inflammation, contributing to their progression and pathogenesis. However, somatic mutagenesis, a driving force in cancer development, has not been generally considered as an important factor in cardiovascular disease pathology. Recent studies demonstrated that during normal aging, somatic mutagenesis occurs in blood cells, often resulting in expansion of mutant clones that dominate hematopoiesis at advanced age. This clonal hematopoiesis is primarily associated with mutations in certain leukemia-related driver genes and, being by itself relatively benign, not only increases the risks of subsequent malignant hematopoietic transformation, but, unexpectedly, has a significant impact on progression of atherosclerosis and cardiovascular diseases. In this review, we discuss the phenomenon of clonal hematopoiesis, the most important genes involved in it, its impact on cardiovascular diseases, and relevant aspects of hematopoietic stem cell biology.