Huntington's disease (HD) is a genetically determined neurodegenerative disorder, the onset of which is known to depend upon the length of glutamine-encoding CAG-repeat sequences lying within the Huntingtin (HTT) gene [1]. Humans may develop the disease if they have more than 36 repeats and disease onset usually starts during mid-life. An inverse relationship has been shown between CAG repeat length and age of onset in HD [2]–[5]. Additionally, somatic instability in human cortex has recently been shown to be a good predictor of disease onset [6]. Children with 108–256 CAG repeats are reported to show disease onset from one and a half years to six years of age [7].

Trinucleotide repeat (TNR) instability varies between organs in a variety of neurodegenerative disorders which are caused by expansion of CAG repeats in a coding gene, with the greatest instability observed in the brain [8]–[11]. In HD, striatum tissue shows the most severe neuropathology, followed by cortex. CAG length expansion is correlated with neuropathology and probably precedes the onset of symptoms [12]. The CAG repeat length is unstable in most cell types of the brain, but neurons tend to show the greatest mutation lengths in both humans and mice [13]–[15]. Meanwhile, minimal expansion is considered to occur in many other somatic tissues.

TNR sequences may form slipped strands during replication or repair, creating loops or hairpins, which protrude from the DNA duplex [16].

In the earliest model for repeat expansion the DNA polymerase forms slip-outs on the nascent strand leading to small-scale repeat expansion in repetitive sequences [17]. Loops of repeat-containing DNA are believed to cause either expansions or contractions during replication, when the slip-out occurs in the nascent or template strand, respectively [18], [19]. Several models have been suggested to explain TNR expansion during replication, such as folding of the lagging strand template into a hairpin, stalled replication forks and the orientation of the TNR in the genome, as well as the location of the origin of replication, as shown in several experiments in bacteria, yeast and human cells (Reviewed in [20]). More recently, a pertinent role of DNA repair proteins in CAG repeat expansion has been demonstrated in vivo. In particular, deletion of the mismatch repair (MMR) proteins, Msh2 and Msh3 [21]–[24] has been shown to abolish age-dependent somatic CAG repeat expansion in mouse models for HD. MMR has also been shown to be involved in TNR expansion in mouse models of myotonic dystrophy (DM1) [25]–[27]. Furthermore, the age-dependent expansion of TNR sequences in somatic cells was shown to be modified by the base excision repair (BER) 8-oxoguanine DNA glycosylase (Ogg1) in the R6/1 mouse model, demonstrating that there may be a link between oxidative DNA damage and TNR instability [13]. The flap endonuclease 1 (FEN1), which removes 5′-flaps during replication [28] and is involved in long-patch BER [29] is also implicated in expansion. Secondary TNR structures have been shown to inhibit FEN1 activity [30]. In addition to flap endonuclease activity, the EXO [31] and GAP activities of FEN1 have been shown to contribute to the resolution of TNR secondary structures in vitro [32]. Recently, it was shown that the stoichiometry of BER proteins, such as Ogg1, polymerase β and FEN1, may contribute to the tissue-selectivity of somatic HD CAG repeat expansion [33].

Nevertheless, the processes causing this expansion remain poorly understood, particularly in mammalian systems, although the formation of secondary DNA structures within the repeat sequence is thought to underlie the process [34]. Here we present evidence for two distinct modes of somatic expansion identified by the analysis of CAG repeat fragments from 103 HD R6/1 mice; a continuous slight expansion in tail, lung, heart and spleen, and a dramatic periodic expansion in striatum, and cortex, which we also compare to the expansions observed in liver. The continuous expansion process is shown here to conform to a bi-directional, forward-biased model that represents the occurrence of multiple short – tending towards unitary – CAG repeat insertions and deletions, at random moments as the mouse ages. In contrast, the dramatic expansion seen in brain tissues demonstrates a periodicity centred around seven repeats, which correlates with the stochastic insertion of stable TNR segments of consistent length. Meanwhile liver tissue shows a comparable average increase in CAG repeat length to striatum, but with a much weaker inclination to exhibit periodicity, tending more towards a continuum. This suggests either a much less controlled insertion length when compared to expansions in striatum, or that liver tissue undergoes both types of expansion simultaneously. We also present discursive models for these two expansion mechanisms. Identification of these two independent modes of expansion, in particular the tight mechanistic control implicit in the expansions within neuropathologically relevant tissues, increases our understanding of the tissue-dependent progress of HD. This brings us a step closer to inferring the in vivo mechanisms of the molecular components involved, by showing that only a limited selection of the existing models for expansion are able to explain the age-dependent CAG repeat expansions we observe.

Having shown data and analysis to define these two distinct modes of expansion, we place our findings into the context of existing literature, in order to develop reasonable hypothetical models for these two types of expansion.

The continuous expansion we observe shows a progressive increase in CAG tract length, which is comparable with the expansions observed in fibroblasts derived from an adult HD mouse [37]. In the debate regarding the relative roles of replication and repair in TNR instability, these results present an interesting question, since the continuous expansion process occurs in organs containing dividing cells. A process occurring with the previously calculated expectation rate of ∼0.036 events per repeat tract per day on the ∼360 nucleotide CAG segment of the 2.5 gigabase mouse genome, would correlate to ∼250,000 genome-wide events per cell per day. This is well above the upper estimate for daily DNA damage events, making it unlikely that these are the sole initiator of expansions in tail tissue. Several replication-based models for TNR expansion in dividing cells [18], [19] have been proposed. The potential for replication to be entirely responsible for this expansion is considered in detail elsewhere (Figures S7, S8, and Text S1) and is considered to be unlikely, particularly in light of the fact that lymphoma tissues (with necessarily higher replication rates) isolated from several mice showed no increase in TNR instability (Figure S7). Therefore we infer that other mechanisms must also prevail, and propose that slipped strand structures [16], [38] generated by out of register rehybridization of CAG repeats [27] during transcription, or genome maintenance, may spontaneously form unstable loops or cruciforms which may subsequently stabilize by migrating apart. Similar small loops can also be formed by polymerase slippage during replication [17]. We propose that two separate pathways may repair these loops, leading to single repeat expansions or contractions (Figure S8). Further research is needed to resolve the specific origin of this mode of expansion.

A hypothesis for periodic expansion is also presented (Figure S9). The previously calculated periodic expansion probability of 0.018 events per repeat tract per day would correlate to ∼100,000 genome-wide events per cell per day. This lies in the vicinity of upper estimates for accumulated oxidative DNA damage [39]. It is therefore possible that DNA damage contributes as catalyst for periodic expansions in brain tissues. However, oxidative damage is not sufficient to trigger somatic instability [33]. Of particular interest here, are the potential molecular components that could repeatedly generate a regularly sized repeat insertion that is dominantly seven repeats in length. We have therefore chosen to briefly review the relevant literature in search of further insight.

Theory suggests that CAG flaps ranging from 4 to 16 triplet repeats in length can form thermodynamically unstable hairpin structures with an even number of repeats [40]. However, under physiological conditions, 6 triplet repeats have been shown to form hairpins irreversibly [41], [42]. This implies that a progressively generated triplet repeat flap can stabilise into a 6-repeat hairpin at the free end that could be cleaved by FEN1 causing no net expansion [43] (Figure S9). However, the presence of metastable intermediates during flap generation may allow the flap length to increase beyond 6 repeats before a stable structure is formed, thereby producing a hairpin with an overhanging CAG at the 5′-end. This free CAG repeat can hybridise back to the DNA duplex, and it has been shown by Liu et al. [31] that such hybridisation facilitates bubble formation followed by ligation and expansion. In a few instances, two or even three free CAG repeats in the 5′-end of the hairpin would produce a periodicity of eight or nine CAG repeat steps. In addition, the size of the hairpin could vary with a few repeats. However, this occurs more rarely, as observed in Figure 4C. After gap filling and ligation, a loop of excess CAG repeats in one strand would be produced (Figure S9) that can be recognized by the MMR complex Msh2-Msh3 with high affinity [44]. This binding might further stabilize the CAG loop and additionally explain why Msh2-Msh3 is necessary for large expansions to occur in striatum [21]–[24], although the role of MMR in causing TNR expansion is not understood. Moreover, Msh2-Msh3 function ceases due to impaired ATPase activity on loops exceeding 16 nucleotides in lengths [45] - probably due to the presence of A•A mispaired bases in the loop [22] - which may explain why the MMR system fails to repair longer extraneous CAG loops. Subsequently, a nick generated on the strand opposite to the loop could result in faulty repair of the CTG strand along the CAG slip-out, causing expansion by a repair process independent of MMR [46] (Figure S9). Thus, oxidative damage on the CTG strand could result in base excision repair (BER) and Ogg1 moderated expansion, which is also in concordance with the proposed ‘toxic oxidation cycle’ by Kovtun et al., 2007 [13]. We therefore propose that a coincidental cooperation can occur between MMR and BER in cases where a long CAG flap is able to stabilize itself, which can form the basis of the consistent periodic insertion we observe.

It should be pointed out that it is possible that other stabilized structures, such as loop-outs or a stabilizing interaction with one of the many proteins and complexes that are in contact with the DNA may also serve to cause the observed periodicity. The majority of the potentially applicable models are covered in the literature [13], [24], [27], [34]. Further work is necessary to resolve this completely.

Cell proliferation in neurons and glial cells has been observed in the subependymal layer adjacent to the caudate nucleus in human HD brains [47]. However, polymerase slippage usually forms small expansions [17], and the repetitive uniformity of the periodic expansion makes it unlikely that polymerase slippage is responsible for the dramatic expansions seen in cortex and striatum. Furthermore, the lack of periodically spaced peaks containing fewer repeats than the mice were born with – as would be expected from a bi-directional process - means that 7-repeat hairpin-based contraction events occur either at a negligible rate in comparison to expansions, or do not occur at all. During the 18 week period, tail-type expansion events are not evident in striatum since they would obfuscate latter periodic peaks (Figure S6). Therefore, the two expansion mechanisms seem to be either entirely independent – not occurring simultaneously in the same cell – or that if they do share common elements, they progress along mutually exclusive pathways. However, it is important to notice that both expansion mechanisms must be dependent on proteins from the MMR complex, since expansions are eliminated in all tissues in either Msh2 or Msh3 nulls in HD [21]–[24], and also in DM1 [25], [26]. To date, we have not managed to define the individual cell types that are specific to these expansion mechanisms. It will be of great interest to compare the expansions observed in animal models to those in cultured HD mouse fibroblasts, as a way of identifying the cell specificity of these modes of expansion.

The liver may be particularly interesting in this regard, since this tissue potentially exhibits a mix of both types of expansion mechanisms. This could be attributed to different modes of expansion occurring in different cell types. Indeed, instability of the DM1 CTG•CAG repeat is known to occur in liver hepatocytes with high DNA ploidy [48].

The question also arises as to why FEN1 did not influence CAG repeat expansion in the organs tested. One might expect a difference, since a recent in vitro study has shown that FEN1, together with long-patch BER of long repeat sequences by polymerase β, promotes expansion by facilitating the ligation of hairpins formed by strand slippage [49]. However, FEN1 flap activity has shown to be circumstantial, with much lower activity in the striatum than in the cerebellum of R6/1 mice [33]. In yeast the capture of flap structures by FEN1, rather than the endonuclease activity, is the most important function of FEN1 in preventing TNR expansion [50]. EXO- and GAP activity of FEN1 are also involved in in vitro triplet repeat expansion in yeast [32], and these activities are probably not influenced by the Fen1^E160D/E160D mutation. Therefore, it seems that the 20% endonuclease activity [36] of the Fen1^E160D/E160D mutation does not affect the rate of CAG repeat expansions. In concordance with our finding Fen1 did not control instability of (CTG)_n*(CAG)_n repeats in a knock-in mouse model for DM1 [51].

It is worth considering briefly why this periodicity has not been described before, since there are numerous potential reasons. One possibility could be that the mice used in the present study exhibit more instability due to environmental factors [52] or genetic background [53]. Perhaps the periodic signal becomes more disperse in older mice used elsewhere; degrading the quality of the data, and that the age-range, as well as the relatively long starting CAG lengths, of our samples is optimal for observing this periodicity. Another possibility is simply that later versions of the GeneMapper system are more sensitive, in comparison to the GeneScan method applied in some older studies, allowing us to see more detail. While periodicity has not featured in other studies of similar tissues and disease models, it is difficult to state unequivocally that it was unobservable in their data. The small volume of data presented in articles, uncertainty over the precise PCR conditions used and the simple fact that periodicity was not the focus of these investigations can be sufficient cause for this phenomenon to have been previously overlooked. There is some variability among replicate striatum samples as shown in Figure S10. This could be explained by sampling error or polymerase slippage in early PCR cycles. Sampling error is however unlikely to be the reason behind the periodicity as explained in Figure S12 and Text S2.

So far, we have only studied periodicity in the R6/1 mouse model and without specific studies of other HD CAG mice models the generality of our data is unknown. Yet, the R6/1 transgenic mouse is a widely accepted and commonly used model for human HD that exhibits a progressive neurological phenotype that exhibits many of the features of HD [54]. HD CAG repeat instability has shown to be similar in humans and mice, with the longest expansion lengths occurring in striatum, followed by cortex, and little expansion in cerebellum and most other tissues [8], [14], [35]. In addition, the HD CAG repeat length appears to be expand most in neuronal cells rather than glial cells in both species [14], [15]. Due to the long starting CAG repeat length in the transgenic mouse, the model may be most relevant as a model for juvenile HD. Given the stated similarities, there are grounds to suspect that the mechanisms of expansion are identical in mice and humans, only occurring at an accelerated pace in the mouse on account of the long repeat tract. In this case, the mouse model would function as a good model for human cases with mid-life age of onset as well. However, periodicity has not previously been reported in HD patients. A possible explanation could be that the repeat length in the R6/1 mouse is longer than the repeat length that has been analyzed in any HD human tissue with regard to somatic instability. The genomic localization of the randomly integrated HD gene fragment in R6/1 mice might modulate the CAG stability. Furthermore, CAG instability in HD patient brain cannot be analyzed at an early time-point that would allow for a direct comparison to the R6/1 data.

Importantly, CAG repeat expansion in human cortex is associated with an earlier age of disease onset, in addition to the role of the constitutional CAG repeat length [6]. This implies that there are disease modifiers that influence somatic instability, and conversely, factors that determine somatic instability which may modify disease pathogenesis. It is therefore critical to understand the mechanism of the different expansion processes and the factors involved.

To summarize, we present two different mechanisms of somatic CAG repeat expansion; a continuous bi-directional expansion in tail, lung, heart and spleen tissues, and a dramatic periodic expansion centred around 7-repeat insertions in striatum and cortex. Further experiments are needed to determine whether the 7-repeat step-size is independent of CAG tract length and species and it remains to be shown whether these two models can explain the expansions observed in other brain tissues and organs, as well as in humans. Nevertheless, these results provide significant new insights into in vivo expansion mechanisms, which may also be relevant to other triplet repeat disorders.