Measuring genome instability in aging - a mini-review

Abstract

Background: There is mounting evidence for an age-dependent accumulation of somatic mutations as a result of the inherent imperfection of DNA replication and repair. A possible age-related decline in genome maintenance systems may exacerbate this age-related loss of genome integrity. A review of the current methods of mutation detection is timely in view of the lack of insight as to the magnitude of somatic mutation accumulation, the types of mutations that accumulate, and their functional consequences.

Objective: In this paper we review the current methods for measuring genome instability in organisms during aging or in relation to life span.

Methods: The review is based on established and novel concepts from the existing literature, with some examples from our own laboratory.

Results: Studies using cytogenetic assays and endogenous or transgenic mutation reporter assays provide strong evidence for age-related increases of different types of mutations in animals and humans during aging. This increase in DNA mutations is tissue-specific and also differs between species.

Conclusion: Today, our knowledge of somatic mutation profiles in aging is mainly derived from cytogenetics and the use of endogenous and transgenic mutation reporter assays. The emergence of new approaches, most notably massively parallel sequencing, will give us deeper insight into the nature of spontaneous genome instability and its possible causal relationship to aging and age-related disease.

Fig. 1

Schematic depiction of the main types of DNA damage and corresponding repair pathways. DSB = double-strand break; NER = nucleotide excision repair; BER = base excision repair; HR = homologous recombination; NHEJ = non-homologous end-joining; MMR = mismatch repair; SSB = single-strand break; TLS = translesion synthesis.

Fig. 2

HPRT assay. Mitotically active cells (fibroblasts, mononuclear cells from blood or spleen) are counted and plated in 6-thioguanine-containing medium. After a period of incubation the wells are inspected for growing colonies. Visible colonies are considered to be mutants. The mutant frequency is the ratio between the cloning efficiency in selective medium and in non-selective medium. HPRT-deficient cells are resistant to 6-thioguanine toxicity and positively selected. Each colony can be analyzed for the spectrum of mutations [for details, see 24].

Fig. 3

Example of a transgenic mutation reporter assay. Multiple lacZ-containing plasmids (only five are shown) are integrated in a head-to-tail orientation at one or more chromosomal locations of C57BL/6 mice. The LacZ-plasmids can be retrieved from host genomic DNA (black waved lines) by HindIII (H) digestion and cloned in Escherichia coli. A variety of inactivating mutations, included point mutations (*), small deletions or insertions (doublearrowed lines) and intra- and inter-chromosomal rearrangements (dashed curve), can be detected by positive selection for inactivation of the lacZ gene [for details, see 30, 32]. Ellipse: origin of replication. A = Ampicillin resistance gene. Gray-colored curve: the 5′ end integration site of the LacZ reporter.

Fig. 4

Single-cell mutation analysis by massively parallel sequencing. a The A→G mutation of interest (red check mark in one of the cells in the whole tissue) cannot be distinguished from the sequencing errors. b When sequencing the genome of the single cell containing the mutation (red check mark in the single cell) after whole genome amplification, the A→G mutation can now be distinguished from the sequencing errors because it shows up in approximately 50% of the reads (box), corresponding to one allele [42].