Mouse tissue culture models of unstable triplet repeats.
ABSTRACT Once into the expanded disease-associated range, trinucleotide repeat alleles become dramatically unstable in the germline and in somatic cells. The molecular mechanism(s) that underlie this unique form of dynamic mutation are poorly understood. Numerous transgenic mouse models of unstable trinucleotide repeats, which reconstitute the dynamic nature of somatic mosaicism observed in humans, have been generated. Given their easy accessibility, tissues from these mice can be collected to establish homogenous cell culture models of trinucleotide repeat dynamics. This chapter describes how such cultures can be established and maintained. Such in vitro systems may be useful to study relevant biological questions concerning fundamental triplet repeat metabolism. In particular, monitoring of repeat stability in cells growing under controlled conditions could help to clarify the relationship among the accumulation of repeat length variation, cell division rates, and DNA replication.
SourceAvailable from: Mario Gomes-Pereira[Show abstract] [Hide abstract]
ABSTRACT: A mounting number of inherited human disorders, including Huntington disease, myotonic dystrophy, fragile X syndrome, Friedreich ataxia and several spinocerebellar ataxias, have been associated with the expansion of unstable simple sequence DNA repeats. Despite a similar genetic basis, pathogenesis in these disorders is mediated by a variety of both loss and gain of function pathways. Thus, therapies targeted at downstream pathology are likely to be disease specific. Characteristically, disease-associated expanded alleles in these disorders are highly unstable in the germline and somatic cells, with a tendency towards further expansion. Whereas germline expansion accounts for the phenomenon of anticipation, tissue-specific, age-dependent somatic expansion may contribute towards the tissue-specificity and progressive nature of the symptoms. Thus, somatic expansion presents as a novel therapeutic target in these disorders. Suppression of somatic expansion should be therapeutically beneficial, whilst reductions in repeat length could be curative. It is well established that both cis- and trans-acting genetic modifiers play key roles in the control of repeat dynamics. Importantly, recent data have revealed that expanded CAG.CTG repeats are also sensitive to a variety of trans-acting chemical modifiers. These data provide an exciting proof of principle that drug induced suppression of somatic expansion might indeed be feasible. Moreover, as our understanding of the mechanism of expansion is refined more rational approaches to chemical intervention in the expansion pathway can be envisioned. For instance, the demonstration that expansion of CAG.CTG repeats is dependent on the Msh2, Msh3 and Pms2 genes, highlights components of the DNA mismatch repair pathway as therapeutic targets. In addition to potential therapeutic applications, the response of expanded simple repeats to genotoxic assault suggests such sequences could also have utility as bio-monitors of environmentally induced genetic damage in the soma.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2006; 598(1-2):15-34. DOI:10.1016/j.mrfmmm.2006.01.011 · 4.44 Impact Factor
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ABSTRACT: Although cataract is a characteristic feature of myotonic dystrophy type 1 (DM1), little is known of the underlying mechanisms. We generated four lens epithelial cell lines derived from DM1 cataracts and two from age-matched, non-DM cataracts. Small-pool PCR revealed typical large triplet repeat expansions in the DM1 cells. Furthermore, real-time PCR analysis showed reduced SIX5 expression and increased expression of the Ca(2+)-activated K(+) channel SK3 in the DM1 cells. These cells also exhibited longer population doubling times which did not arise through reduced proliferation, but rather increased cell death as shown by increased release of lactate dehydrogenase (LDH). Using (86)Rb(+) as a tracer for K(+), we found no difference in the resting K(+) influx or efflux kinetics. In all cases, the ouabain sensitive component of the influx contributed approximately 50% of the total. However, stimulating internal Ca(2+) by exposure to ionomycin not only caused greater stimulation of K(+) ((86)Rb) efflux in the DM1 cells but also induced a higher rate of cell death (LDH assay). Since both the hyper-stimulation of K(+) efflux and cell death were reduced by the highly specific SK inhibitor apamin, we suggest that increased expression of SK3 has a critical role in the increased Ca(2+)-induced fragility in DM1 cells. The present data, therefore, both help explain the lower epithelial cell density previously observed in DM1 cataracts and provide general insights into mechanisms underlying the fragility of other DM1-affected tissues.Human Molecular Genetics 01/2007; 15(24):3559-68. DOI:10.1093/hmg/ddl432 · 6.68 Impact Factor