Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates

Abstract

The expansion of CAG.CTG trinucleotide repeats has been associated with an increasing number of human diseases. Once into the expanded disease-associated range, the repeats become dramatically unstable in the germline and also throughout the soma. Instability is expansion-biased, contributing towards the unusual genetics, and most likely the tissue-specificity and progressive nature of the symptoms. Such expansions constitute a unique form of dynamic mutation whose mechanism is poorly understood. It is generally assumed that repeat length changes arise via replication slippage, yet no direct evidence exists to support this hypothesis in a mammalian system. We have previously generated transgenic mouse models of unstable CAG.CTG repeats that reconstitute the dynamic nature of somatic mosaicism observed in humans. We have now used tissues from these mice to establish in vitro cell cultures. Monitoring of repeat stability in these cells has revealed the progressive accumulation of larger alleles as a result of repeat length changes in vitro, as confirmed by single cell cloning. We also observed the selection of cells carrying longer repeats during the first few passages of the cultures and frequent additional selective sweeps at later stages. The highest levels of instability were observed in cultured kidney cells, whereas the transgene remained relatively stable in eye cells and very stable in lung cells, paralleling the previous in vivo observations. No correlation between repeat instability and the cell proliferation rate was found, rejecting a simple association between length change mutations and cell division, and confirming a role for additional cell-type specific factors.

Unusual tumors associated with the hereditary nonpolyposis colorectal cancer syndrome

“…24 Cell cultures derived from the eyes, kidneys, and lungs from these transgenic mice demonstrated different levels of repeat instability, independent of cell proliferation rate. 23 Similar observations have been made in humans with myotonic dystrophy type 1, as larger repeat lengths are consistently observed in muscle compared to blood DNA. [25][26][27] Although we favor the possibility of tissue-specific factors influencing microsatellite instability, there is currently no experimental evidence to support this in HNPCC.…”

“…A third possible explanation for the decreased microsatellite instability in extra-colonic cancers is that cis and/or trans acting tissue-specific factors, along with defects in MLH1 or MSH2, play a role in determining instability of microsatellites. 23 Evidence that supports this explanation comes from transgenic mouse models of unstable CAGKCTG repeats, which are expanded in diseases such as myotonic dystrophy type 1, Huntington's disease, fragile X syndrome, Friedreich's ataxia, and spinocerebellar ataxias. 24 Cell cultures derived from the eyes, kidneys, and lungs from these transgenic mice demonstrated different levels of repeat instability, independent of cell proliferation rate.…”

Modelling and inference reveal nonlinear length-dependent suppression of somatic instability for small disease associated alleles in myotonic dystrophy type 1 and Huntington disease

“…The precise mechanism by which somatic mosaicism arises remains unknown, but it is assumed that it must involve the misalignment of repeats to generate small loops, hairpins or other alternative non-B-DNA structures [1,17,18]. Although the repeats may be unstable during replication, the lack of a correlation between tissue-specific somatic mosaicism and cell proliferation and the accumulation of high levels of somatic mosaicism in post-mitotic tissues such as brain and muscle [11,12,20,[26][27][28][29], suggest the primary mechanism is cell division independent. Evidence from transgenic mouse models also implicates a major role for several of the DNA mismatch repair proteins [30 -33], and inappropriate DNA mismatch repair of alternative structures presents as an attractive mechanism [34].…”

Hijacking of the mismatch repair system to cause CAG expansion and cell death in neurodegenerative disease

“…Thus, their role in causing expansion is difficult to test. Further, tests of expansion among cell types have not always painted a consistent picture due to the fact that the effects of strand breaks cannot be separated from the effects of replication in proliferating cells [67][68][69][70][71][72][78][79][80]. However, the use of transgenic animals has been particularly informative [70,[80][81][82][83][84][85][86].…”