Understanding the factors that modify disease phenotypes in HD patients provides a critical route to therapeutic targets. The age of clinical motor onset, a well-defined phenotypic milestone, that reflects the rate of the HD pathogenic process, is strongly dependent on HD
CAG repeat length, but is also modified by other environmental and genetic factors, some of which have been identified (10
). Intriguingly, the HD
CAG repeat is somatically unstable, progressively increasing in length over time, particularly the brain regions (striatum and cortex) that succumb earliest to disease pathogenesis (25
). Given the dependence of onset age on HD
CAG repeat length, this raises the hypothesis that somatic HD
CAG repeat instability in these tissues accelerates the pathogenic process. Here, we present the first study to explore this hypothesis in HD individuals by rigorously quantifying, at the single molecule level, HD
CAG repeat lengths in a large number of patient cortex samples, and correlating the level of somatic instability in cortex with the age of disease onset. We find that, after controlling for effects of constitutive HD
CAG repeat length on both somatic instability and onset age, somatic instability is significantly associated with onset age, with greater somatic expansions seen with earlier ages of onset. Our findings suggest that factors that contribute to differences in somatic instability between individuals may also be modifiers of disease.
The association between somatic instability and clinical onset does not directly demonstrate that increased somatic instability accelerates the pathogenic process. It is possible that somatic instability is consequence of pathogenesis, as suggested (35
), with greater levels of somatic instability simply reflecting a more rapid pathogenic process in individuals with early onset disease. However, several lines of evidence would argue against this possibility. First, high levels of somatic instability in striatum and cortex are seen in other CAG/CTG repeat disorders, notably spinocerebellar ataxia type 1 (SCA1) and myotonic dystrophy type 1 (DM1) (36
). As the major targets of neurodegeneration in these disorders lie in tissues and brain regions outside striatum and cortex, somatic instability is unlikely to be a consequence of a disease process, but rather to be due to normal tissue-specific factors that are unrelated to disease state. Similarly, greater HD
CAG somatic expansion is not seen in transgenic mouse models that exhibit dramatic phenotypes in response to the C-terminal fragment of the HD
gene compared with Hdh
CAG knock-in mice exhibiting a slow disease course (29
). Furthermore, data from our laboratory demonstrate that accelerating the disease process in Hdh
CAG knock-in mice does not increase somatic instability (J.-M. Lee and V.C. Wheeler, unpublished data).
Rather than instability being a consequence of pathogenesis, data from Hdh
knock-in mice suggest that instability is a modifier of the pathogenic process (31
). When somatic instability in the striatum of HdhQ111
CAG knock-in mice was eliminated by crossing these mice onto genetic backgrounds deficient in mismatch repair genes Msh2
, nuclear mutant huntingtin immunoreactivity in striatal nuclei, a HD
CAG repeat length-dependent presymptomatic phenotype, was delayed (31
). Conversely, deficiency of Msh6
had no effect on somatic instability and did not alter nuclear mutant huntingtin immunoreactivity (32
). These data imply that somatic instability contributes to the HD
CAG pathogenic process in Hdh
knock-in mice. While additional experiments are needed in the mouse to unambiguously demonstrate the role of somatic instability in the disease process, our data showing an inverse correlation between somatic instability in HD patient cortex and age of disease onset are consistent with, and support the hypothesis, that somatic instability contributes to the pathogenic process in HD.
How significant a role might somatic instability play in modulating the HD pathogenic process? It has recently been proposed that somatic expansion of the HD
CAG repeat beyond a certain ‘pathological threshold’ of ~115 CAG repeats is required before overt HD symptoms ensue (38
), i.e. that somatic expansion is necessary
for disease onset. This would predict that, starting at the same constitutive repeat length, individuals with more somatic expansion would reach this threshold earlier than individuals with less somatic expansion, and therefore exhibit earlier disease onset. While our data are consistent with this hypothesis, they cannot distinguish somatic expansion over a threshold as the instigator of disease, as proposed by Kaplan et al
), from somatic instability as modifier of a disease process that would proceed even in its absence. These possibilities may best be addressed by determining the effect of somatic instability on phenotypes in accurate genetic mouse models of HD containing repeat lengths that are below the predicted ‘pathological threshold’.
It is difficult to determine from our study the possible contribution of somatic instability to onset age. As our HD sample included only those individuals displaying phenotypic extremes of young and old onset, the proportion of the variation of onset age that is accounted for by somatic instability is not representative of the HD population as a whole. In addition, as neurons with the longest repeat expansions may be preferentially lost during the disease course (27
), the HD
CAG repeat length distribution in end-stage brain may not be an accurate reflection of that present at disease onset. Therefore, we believe that the association between somatic instability and onset age in our study is likely an underestimate of the true association if somatic instability could be measured in the brain at the time disease onset.
While analyses of HD
CAG repeat lengths in brains of rare presymptomatic HD
gene carriers (27
) provide insight into the extent of somatic instability preceding the onset of symptoms, they do not allow an estimate of the correlation with disease onset. It may, however, be possible to correlate measures of somatic instability in other tissues, such as blood/lymphoblasts, fibroblasts or buccal cells with onset age (34
). Interestingly, as in the present study, analyses of the variation in buccal cell somatic instability between individuals provided evidence for modifiers of instability other than constitutive HD
CAG repeat length (34
). Thus, if an individual's propensity for somatic instability in the brain is reflected in peripheral tissues, it is possible that somatic instability in these tissues could be used as a surrogate for CAG repeat instability in the brain.
It would also be interesting to determine whether somatic instability is a predictor of additional clinical markers of disease, particularly early phenotypes that precede the overt onset of motor symptoms, e.g. cognitive and psychiatric symptoms and cortical changes (40
). The expectation is that any phenotypes that are CAG repeat length-dependent would be modifiable by somatic instability, and would therefore show an association with somatic instability.
In summary, we suggest that somatic instability is a modifier of the HD pathogenic process. This predicts that factors that determine somatic instability in HD patients will also modify disease pathogenesis, and conversely, that disease modifiers may also influence somatic instability. Somatic instability is also predicted to alter disease phenotypes in other trinucleotide repeat disorders in which somatic instability is prevalent in tissues affected by the disorder, notably muscle in DM1 (43
). Profiling somatic repeat instability over a large number of HD patient samples, as we have begun in this HD study, may provide a resource to identify genetic factors that determine somatic instability in humans. Interestingly, we observed inter-individual differences in the instability of the normal HD
CAG repeat, as well as in the mutant HD
CAG repeat (Fig. A). Factors such as Msh2
, found to be important in generating somatic instability of an expanded HD
CAG repeat in mouse models of HD (31
), provide sources of candidate genes predicted to alter both somatic instability and age of onset in HD patients. Thus, with the aim of slowing the pathogenic process that leads to this destructive disease, our data support pursuing somatic instability as a novel and viable therapeutic target.