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. 2007 Jul 23:8:61.
doi: 10.1186/1471-2199-8-61.

Somatic CTG*CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy

Walther J A A van den Broek  1 , Derick G Wansink, Bé Wieringa
Affiliations

Somatic CTG*CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy

Walther J A A van den Broek et al. BMC Mol Biol. .

Abstract

Background: Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG*CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation.

Results: After separation of liver hepatocytes and pancreatic acinar cells in pools with 2n, 4n or 8n DNA, we analyzed CTG*CAG repeat length variation by resolving PCR products on an automated PAGE system. We observed that somatic CTG*CAG repeat expansion in our DM1 mouse model occurred almost uniquely in the fraction of cells with high cell nuclearity and DNA ploidy and aggravated with aging.

Conclusion: Our findings suggest that post-replicative and terminal-differentiation events, coupled to changes in cellular DNA content, form a preconditional state that influences the control of DNA repair or recombination events involved in trinucleotide expansion in liver hepatocytes and pancreatic acinar cells.

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Figures

Figure 1
Figure 1
Effects of aging on the somatic CTG•CAG repeat length distribution in heart, liver and pancreas. Typical CTG•CAG length profiles of genomic DNA isolated from heart (A), liver (B) or pancreas (C) of a knock-in mouse model carrying a humanized DMPK gene with a (CTG•CAG)n repeat [13]. Arrows indicate the position of the progenitor allele determined from analysis of tail DNA at time of weaning. Note the slow but uniform broadening of the profile with slight shift towards greater repeat lengths upon aging in heart, in contrast to the bimodal repeat length distribution in liver and pancreas.
Figure 2
Figure 2
Polyploid cells in liver and pancreas display enhanced somatic instability of the CTG•CAG repeat. (A, B) Liver hepatocytes from a 17-month-old mouse and pancreatic acinar cells from a 21-month-old mouse were isolated, fixed, stained with propidium iodide and FACS purified based on DNA content. Preparative sorting profiles based on PI fluorescence intensity are shown. All 2n hepatocytes were mononuclear, whereas ~80% cells in the 4n and 8n pools of hepatocytes were binuclear (C, inset; typical examples of cells in the sorted 2n and 4n cell pools stained with Giemsa, are shown). Note that pancreas contained only diploid and tetraploid cells (a fluorescent PAS staining of zymogen inclusions established the identity of acinar cells (D, inset)). (C, D) CTG•CAG length profiles of hepatocytes and acinar cells demonstrated much larger average repeat length and almost complete absence of progenitor alleles in cells with 4n DNA (red; liver hepatocytes and acinar cells) and 8n DNA (black; liver hepatocytes only). In the 2n pools obtained from liver and pancreas (blue) only a small percentage of cells contained expanded repeats. Arrows indicate the position of the progenitor allele determined from analysis of tail DNA at time of weaning.
Figure 3
Figure 3
Somatic instability of CTG•CAG repeats in liver hepatocytes of mice containing two humanized DMPK alleles (compound homozygotes). Hepatocytes were isolated from a 14-month-old compound homozygous (CTG•CAG)104/(CTG•CAG)112 and a 16-month-old (CTG•CTG)105/(CTG•CAG)110 mouse and sorted in 2n and 4n cells. PCR profiles of genomic DNA show that in both mice the average CTG•CAG repeat length in the pool of 4n cells (red) is much larger than in the 2n cell pool (blue) and shifted to larger length for both progenitor alleles. Arrows indicate the positions of the two progenitor (CTG•CAG)n alleles in tail DNA at time of weaning.
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