Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Dec 9;6(12):e1001242.
doi: 10.1371/journal.pgen.1001242.

Continuous and periodic expansion of CAG repeats in Huntington's disease R6/1 mice

Linda Møllersen  1 , Alexander D Rowe, Elisabeth Larsen, Torbjørn Rognes, Arne Klungland
Affiliations

Continuous and periodic expansion of CAG repeats in Huntington's disease R6/1 mice

Linda Møllersen et al. PLoS Genet. .

Abstract

Huntington's disease (HD) is one of several neurodegenerative disorders caused by expansion of CAG repeats in a coding gene. Somatic CAG expansion rates in HD vary between organs, and the greatest instability is observed in the brain, correlating with neuropathology. The fundamental mechanisms of somatic CAG repeat instability are poorly understood, but locally formed secondary DNA structures generated during replication and/or repair are believed to underlie triplet repeat expansion. Recent studies in HD mice have demonstrated that mismatch repair (MMR) and base excision repair (BER) proteins are expansion inducing components in brain tissues. This study was designed to simultaneously investigate the rates and modes of expansion in different tissues of HD R6/1 mice in order to further understand the expansion mechanisms in vivo. We demonstrate continuous small expansions in most somatic tissues (exemplified by tail), which bear the signature of many short, probably single-repeat expansions and contractions occurring over time. In contrast, striatum and cortex display a dramatic--and apparently irreversible--periodic expansion. Expansion profiles displaying this kind of periodicity in the expansion process have not previously been reported. These in vivo findings imply that mechanistically distinct expansion processes occur in different tissues.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Fragment analysis in tail, cortex, striatum, and liver.
Representative examples of raw data from CAG-repeat sequences in tail (A), cortex (B) striatum (C) and liver (D) from individual male and female HD mice, aged 10 and 21 weeks are shown (blue) with the tail biopsy from the same 3-week old mouse overlaid (red). All traces demonstrate the increase in mean length of repeat sequences with time and the differing rates of expansion between tissue types. Of particular note is the strong periodicity shown in the older striatum samples. Size standard markers are shown for 118 (solid black arrow) and 138 (open white arrow) CAG repeats respectively.
Figure 2
Figure 2. Slight continuous expansion measured in tail tissue.
Size standard markers, are placed at 118 (solid black arrow) and 138 (open white arrow) CAG repeats. (A) A representative example of raw data from the 3-week biopsy (red) and 21-week sample (blue) are shown for one mouse. (B) Normal distributions are fitted to the data presented in (A), coloured as previously for 3-week (red) and 21-week (blue) samples. The resulting means (μ) and standard deviations (σ) are used to define the temporal change in repeat distribution within the sample, clearly demonstrating an increase in the mean number of repeats between 3 weeks (μ3) and 21 weeks of age (μ21). Likewise, broadening of the distribution is evident from the increase in standard deviation at 3 weeks (σ3) to that at 21 weeks (σ21). (C) Mean values (μ) for repeat lengths from the 3-week (red) and 21-week (blue) tails samples of 59 mice are compiled into a histogram, showing the systematic increase in repeat length with age. (D) Standard deviations of all 59 tail samples at 3-weeks (red) and 21-weeks (blue) are similarly compiled, with the histogram showing age dependent peak broadening. (E) A boxplot of expansions measured in the tails of 59 mice between 3 and 21 weeks of age shows a median expansion of 1.97 CAG repeats.
Figure 3
Figure 3. Age-dependent increase in mean and standard deviation for tail tissue.
(A) Mean versus standard deviation points are plotted for all 103 mice in the data set, with the data divided into 3-week (red), 10-week (blue) and 21-week (green) age groups. To highlight the general trends in the data, the points representing the middle quartiles (by mean value) for each age group are shown with solid circles. The 3-week age group is shown with the weak positively-correlated trend line (red) implying a loose relation between standard deviation and mean value at a fixed age, which we assume to be caused by polymerase errors – during PCR of repeat sequences – which increase with sequence length. This trait is also present in 10-week and 21-week data, with parallel approximate trends shown (dashed) for emphasis. The standard deviations and means for each age group are, however, shown to increase systematically with age, along a trend-line (black arrow) that is completely separate from the PCR-dependent trend. This demonstrates the independence of the age-dependent increase in standard deviation and mean from the PCR induced variation. (B) Monte Carlo simulation of the proposed model mechanism for expansion is shown with a range of parameters for expansion and contraction probabilities pe and pc, to illustrate the change in mean and standard deviation of a distribution from a given starting point, dependent upon the relative expansion and contraction probabilities. It is clear from the results that only the combination of probabilities calculated from tail data can combine to generate the measured simultaneous change in mean and standard deviation.
Figure 4
Figure 4. Periodic expansions in striatum and cortex.
(A) Fragment analysis curves from 3-week tail, 10- and 21-week striatum are shown sequentially, with fitted normal distributions (black) overlaid. The sum of all fits (red) demonstrates the curve-fit accuracy. Mean values (μa etc) for each fitted peak are shown. Mean μa coincides with the mean of the corresponding 3-week tail at all ages. This 21-week striatum sample is best-fit by six consecutive normal distributions. Periodicity is clear from the regularity of the intervals between consecutive means. (B) Multiple peaks fitted to another 21-week striatum dataset are shown, with the areas under each fitted distribution (Aa .. Ad), the mean values and the separations between the four peaks (S1 .. S3). The area of a peak represents the proportion of tissue containing the measured mean number of repeats, thus separation values represent a step-wise expansion from the previous mean. The age-dependent propagation of peaks with higher means, as seen in (A), is due to the stochastic insertion of short repeat sequences, of consistent length, into the CAG tracts of individual cells, which, over time, generates the observed periodicity. For analysis purposes, the sum of all areas (Aa,b,c,d) was rescaled to 10,000 (the approximate number of cells forming a sample), allowing the number and length of insertion events to be estimated. (C) A histogram of insertion lengths for all expansion events measured in 69 separate striatum samples is shown (blue). Both mean and median values of the distribution point to a dominant insertion length of seven CAG repeats. The separate contributions from 10-week (green) and 21-week (red) data are also shown. Inset figure shows a similar result for cortex, with the insertion length distributed at 7 repeats, despite the smaller number of insertion events observed in total.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell. 1993;72:971–983. - PubMed
    1. Andrew SE, Goldberg YP, Kremer B, Telenius H, Theilmann J, et al. The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease. Nat Genet. 1993;4:398–403. - PubMed
    1. Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, et al. Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat Genet. 1993;4:387–392. - PubMed
    1. Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, et al. Relationship between trinucleotide repeat expansion and phenotypic variation in Huntington's disease. Nat Genet. 1993;4:393–397. - PubMed
    1. Stine OC, Pleasant N, Franz ML, Abbott MH, Folstein SE, et al. Correlation between the onset age of Huntington's disease and length of the trinucleotide repeat in IT-15. Hum Mol Genet. 1993;2:1547–1549. - PubMed

Publication types