Genome instability: a conserved mechanism of ageing?

Abstract

DNA is the carrier of genetic information and the primary template from which all cellular information is ultimately derived. Changes in the DNA information content through mutation generate diversity for evolution through natural selection but are also a source of deleterious effects. It has since long been hypothesized that mutation accumulation in somatic cells of multicellular organisms could causally contribute to age-related cellular degeneration and death. Assays to detect different types of mutations, from base substitutions to large chromosomal aberrations, have been developed and show unequivocally that mutations accumulate in different tissues and cell types of ageing humans and animals. More recently, next-generation sequencing-based methods have been developed to accurately determine the complete landscape of base substitution mutations in single cells. The first results show that the somatic mutation rate is much higher than the germline mutation rate and that base substitution loads in somatic cells are high enough to potentially affect cellular function.

Keywords: DNA mutation; DNA repair; Somatic mutation; ageing.

Figure 1. DNA damage, mutations and ageing

Schematic depiction of DNA damage and mutations in somatic tissues of ageing organisms and their possible consequences.

Figure 2. Somatic mutations accumulate with age in four different tissues

Using a transgenic mouse model harboring chromosomally integrated plasmids containing the lacZ reporter gene that can be excised and transferred into E. coli to select for mutants that inactivate the lacZ-encoded β-galactosidase, mutation frequency (y-axis) was determined as a function of the age of the animals. Each determination point is the average of at least five individual animals. Data were redrawn from [33] and [34].

Figure 3. Single-cell analysis is required for detecting low-abundant, random mutations

The T > G mutation (red dot ) can only be distinguished from sequencing errors after single-cell, whole genome amplification (WGA) when it shows up in ~50% of the reads (yellow; one mutated allele). Polymorphic variants (SNPs; blue) are also observed in the unamplified control DNA extracted from the bulk cell population. SNPs are variants between the genome of our cells and the reference genome. They are electronically discarded (after [65]).

Figure 4

Schematic representation of whole genome sequence analysis of mutations in single human, primary fibroblasts after amplification and in unamplified clones from single fibroblasts taken from the same population.

Figure 5. Frequency of somatic mutations in unamplified clones and amplified single cells

Clones and MDA amplicons derived from single human dermal fibroblasts were sequenced and the variants found compared with those in the bulk population to determine the somatic mutation frequency. There was no significant difference (P=0.76, Wilcoxon test) in the frequency of mutations observed, indicating that the single cell amplification protocol provides an accurate estimate of the somatic mutation frequency. The right-hand axis indicates the number of mutations per cell after adjustment for coverage. Error bars indicate 88% confidence intervals. From Milholland et al. [51].

Figure 6. Germline and somatic mutations in humans and mice

In humans and mice, germline mutation frequencies were measured by sequencing trios of parents and offspring, while somatic mutation frequencies were determined by sequencing single cells. Mutation rates were determined by dividing mutation frequencies observed by the estimated number of mitoses per generation. The heights of the bars reflect the median mutation rate (n=12, 8, 10, and 5) on a logarithmic scale. Error bars indicate +/−1 standard deviation. The somatic mutation rate was nearly two orders of magnitude higher than the germline mutation rate in both species; in mice, both the germline and somatic mutation rates were several times higher than their human equivalents. All differences were statistically significant (P<0.01, Wilcoxon test; after [51]).