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. 2011;6(8):e23647.
doi: 10.1371/journal.pone.0023647. Epub 2011 Aug 29.

Quantification of age-dependent somatic CAG repeat instability in Hdh CAG knock-in mice reveals different expansion dynamics in striatum and liver

Jong-Min Lee  1 , Ricardo Mouro Pinto, Tammy Gillis, Jason C St Claire, Vanessa C Wheeler
Affiliations

Quantification of age-dependent somatic CAG repeat instability in Hdh CAG knock-in mice reveals different expansion dynamics in striatum and liver

Jong-Min Lee et al. PLoS One. 2011.

Abstract

Background: Age at onset of Huntington's disease (HD) is largely determined by the CAG trinucleotide repeat length in the HTT gene. Importantly, the CAG repeat undergoes tissue-specific somatic instability, prevalent in brain regions that are disease targets, suggesting a potential role for somatic CAG repeat instability in modifying HD pathogenesis. Thus, understanding underlying mechanisms of somatic CAG repeat instability may lead to discoveries of novel therapeutics for HD. Investigation of the dynamics of the CAG repeat size changes over time may provide insights into the mechanisms underlying CAG repeat instability.

Methodology/principal findings: To understand how the HTT CAG repeat length changes over time, we quantified somatic instability of the CAG repeat in Huntington's disease CAG knock-in mice from 2-16 months of age in liver, striatum, spleen and tail. The HTT CAG repeat in spleen and tail was very stable, but that in liver and striatum expanded over time at an average rate of one CAG per month. Interestingly, the patterns of repeat instability were different between liver and striatum. Unstable CAG repeats in liver repeatedly gained similar sizes of additional CAG repeats (approximately two CAGs per month), maintaining a distinct population of unstable repeats. In contrast, unstable CAG repeats in striatum gained additional repeats with different sizes resulting in broadly distributed unstable CAG repeats. Expanded CAG repeats in the liver were highly enriched in polyploid hepatocytes, suggesting that the pattern of liver instability may reflect the restriction of the unstable repeats to a unique cell type.

Conclusions/significance: Our results are consistent with repeat expansion occurring as a consequence of recurrent small repeat insertions that differ in different tissues. Investigation of the specific mechanisms that underlie liver and striatal instability will contribute to our understanding of the relationship between instability and disease and the means to intervene in this process.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. GeneMapper analysis of HdhQ111 CAG repeats shows pronounced age-dependent repeat instabilities in striatum and liver.
Genomic DNA from liver, striatum, spleen and tail of HdhQ111/+ mice (CD1 background) was used for PCR amplification of the HTT CAG repeat followed by GeneMapper analysis. Five time points were used: 2 months (5 mice); 5 months (5 mice); 9 months (5 mice); 12 months (3 mice); 16 months (5 mice). Representative GeneMapper traces are shown.
Figure 2
Figure 2. Age-dependent increase of somatic instability in liver and striatum suggests near linear increases in instability in these tissues.
Somatic repeat instability was quantified from GeneMapper traces by determining an instability index (Methods) for striatum, liver, spleen and tail of each HdhQ111 /+ mouse (CD1 background) at each time point: 2 months (5 mice); 5 months (5 mice); 9 months (5 mice); 12 months (3 mice); 16 months (5 mice). Instability index was plotted against age. Constitutive CAG repeat lengths are color-coded. Circle, liver; triangle, striatum; square, spleen; and diamond, tail.
Figure 3
Figure 3. Quantification of the different patterns of somatic repeat instability in liver and striatum.
To capture different patterns of repeat instability in liver and striatum, two additional measurements were made. (A) Repeats in liver showed a characteristic bimodal distribution and to quantify this, the distance between the modes of the constitutive and somatically expanded repeats was determined. The mode-mode distance was plotted at each time-point: 5 months (3 mice); 9 months (4 mice); 12 months (3 mice); 16 months (5 mice). All mice at 2 months, two mice at 5 months, and one mouse at 9 months did not show a distinct second mode. Two mice at 5 months showed the same distances between the two modes. (B) In striatum, the mode of the somatically expanded repeats was not distinct. Therefore, we measured the distance between the constitutive repeat mode and the longest repeat after background correction, and compared these to those of liver at each time point: 2 months (5 mice); 5 months (5 mice); 9 months (5 mice); 12 months (3 mice); 16 months (5 mice). Blue circle, liver; red triangle, striatum. Red line represents relative peak threshold based on 10% threshold factor that was used for background correction.
Figure 4
Figure 4. Hepatocytes are enriched for unstable HTT CAG repeats particularly when in polyploidy states.
(A) GeneMapper traces of PCR-amplified HTT CAG repeats from isolated hepatocytes (top panel), whole liver (middle panel) and tail DNA (bottom panel) from a 4 month old HdhQ111/+ mouse (FVB/N background, constitutive CAG142) were shown. Isolated hepatocytes show an enrichment of unstable CAG repeats when compared to a whole liver preparation. (B, C and D) Hepatocytes isolated from a 9 month old HdhQ111/+ mouse (C57BL/6J background, constitutive CAG 131) were fixed, stained with propidium iodide and FACS sorted based on DNA content into 2N, 4N and 8N hepatocytes pools. (B) FACS analysis indicated that approximately 95% of the hepatocytes were in polyploid states (4N or 8N). X-axis and Y-axis represent DNA content and cell count, respectively. (C) GeneMapper traces of PCR-amplified HTT CAG repeat of unsorted hepatocytes (top panel: mixture of 2N, 4N and 8N hepatocytes) and FACS sorted 2N, 4N and 8N hepatocytes pools revealed that stable CAG repeats are almost exclusively present in the 2N hepatocytes pool and that unstable CAG repeats are strongly enriched in both 4N and 8N hepatocytes pools. (D) Cytological analysis of the FACS sorted hepatocytes confirmed a decline of mononuclearity with increasing nuclear content . However, due to the lack of a clear boundary between 2N and 4N cells, the FACS sorted 2N hepatocytes pool was susceptible to contamination from the 4N cells, resulting in the presence of a significant proportion of binucleated hepatocytes (23%) in the 2N hepatocyte pool. It is also important to note that mononucleated 2N pools may contain mononucleated 4N cells.
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