As can be gathered from chapters in this volume, research is beginning to clarify the relationship between nucleotide expansions and their consequences. Perhaps not surprisingly, these consequences depend on some combination of the properties of the repeat itself, its location in the affected gene, and the function of that gene. When the repeat is located in an open reading frame, the relationship between expansion and disease pathology is superficially quite straightforward; nucleotide expansion causes an increase in the length of a run of a particular amino acid, which in all instances to date has been glutamine, and the resulting polyglutamine (polyQ) stretch is toxic. However, the basis of the toxicity of the polyQ tract remains the subject of vigorous debate and, as discussed by Friedman et al. in this volume, mechanisms still under consideration include mitochondrial dysfunction, transcriptional dysregulation, axonal transport defects, and cytoskeletal abnormalities. The role of protein context in toxicity is becoming increasingly apparent, and in the case of SCA6 a lack of agreement exists as to how much of the pathology is due to polyQ toxicity and how much results from loss of normal protein function (Frontali, this volume).
The effects of expansion of repeats that are located outside the open reading frame seem to be remarkably varied. Some expansions are thought to constitute loss-of-function mutations, while others represent a gain of function. Loss-of-function expansions include those in which the primary promoter structure is disrupted, repeat-induced epigenetic changes lead to gene silencing, or blocks are formed to either transcription or translation. While definitive proof of the actual disease mechanism is often lacking, in many instances available evidence implicates unusual mechanisms. For example, the transcription block that has been suggested to cause Friedreich ataxia (FA or FRDA) may involve the formation of an unusual DNA structure, such as a triplex or "sticky DNA", that impedes RNA polymerase (Pandolfo, this volume). All the known gain-of-function expansions involve a novel disease paradigm: RNA-mediated pathology (Teng-umnuay and Swanson, and Tassone and Hagerman, this volume), which has just recently begun to be elucidated.
It has also been appreciated recently that expansion can have different consequences depending on the number of repeats. For example, as discussed by Tassone and Hagerman in this volume, carriers of FMR1 alleles with 59200 repeats are at risk for fragile X associated tremor and ataxia syndrome (FXTAS) and fragile X associated premature ovarian failure (FXPOF). However, carriers of alleles with more than 200 repeats have a completely different disorder, fragile X mental retardation syndrome (FXS). FXTAS and FXPOF are thought to represent gain-of-function disorders resulting from the effects of the expanded repeats in the transcript, while FXS results from loss-of-
gene-function, specifically the absence of FMRP, the protein product of FMR1 due to silencing of the gene. FXTAS has been suggested to be a functional laminopathy resulting from sequestering of lamin A/C (Arocena et al. 2005); however, it remains to be seen whether such an effect accounts for all FXTAS symptoms and whether the same mechanism is responsible for FXPOF. The mechanism of gene silencing in FXS is also unknown. Interestingly, the expanded FMR1 alleles are transcribed in early embryogenesis and the r(CGG) repeats form RNA hairpins that are substrates for the ribonuclease Dicer (Handa et al. 2003). This raises the possibility (Handa et al. 2003; Jin et al. 2004) that silencing of FMR1 may be mediated in some instances through the RNA interference pathway (Matzke and Birchler 2005).
RNA-mediated pathology is also thought to be responsible for most cases of myotonic dystrophy (DM1) (Teng-umnuay and Swanson, this volume). However, in congenital DM1, a very severe form of DM1 seen only when the repeat tract is extremely large, another novel disease mechanism may contribute to disease symptoms. In most cases of DM1, the repeats are hete-rochromatinized owing to the antisense effect of a bidirectional promoter of the downstream SIX5 gene. Heterochromatin spreading is blocked by a CTCF-dependent insulator element. In congenital DM1, CTCF binding is lost, resulting in heterochromatin spreading into adjacent genes (Cho et al. 2005). The heterochromatization may affect the expression of adjacent genes, thereby accounting for some of the unusual features of this form of DM1. The cases of FXS, FXTAS, and FXPOF and of the different types of DM1 raise the possibility that other repeat expansions may also have more than one pathological mechanism.
While the broad outlines of many nucleotide repeat disorders are beginning to emerge, many of them are still bereft of molecular details. Large gaps in our knowledge are most apparent for particular diseases. The relative rarity of spinocerebellar ataxias (SCA) types 8 and 10, and the limited access to affected tissues has slowed progress toward elucidation of the disease mechanism (Dick et al., and Lin and Ashizawa, this volume). In the case of Huntington disease-like 2 (HDL2) and SCA12, both of which involve expansions of the repeat CAG ■ CTG, whether the repeats are translated is not even known (Margolis et al., this volume). While it is possible that the repeat falls into an open reading frame in a subset of alternative transcripts, it is unlikely that a polyQ tract is responsible for either disorder. It remains possible in the case of HDL2 that a polyalanine- or a polyleucine-containing protein is involved. Such amino acid tracts are toxic in cell models and a polyala-nine tract has been shown to be responsible for oculopharyngeal muscular dystrophy (Brais et al. 1998). RNA toxicity also remains a viable explanation for both disorders, although in the case of SCA12 it would probably have to involve CAG-RNA rather than CUG-RNA. If this turns out to be the case, it would represent the first disease resulting from CAG-RNA-mediated pathology. However, the muscleblind proteins thought to be involved in the pathology responsible for DM1 and DM2 bind to RNA containing CHHG and CHG repeats, where H is A, U, or C (Kino et al. 2004). Thus, it is possible that the net effect of the SCA12 repeat is very similar to that of DM1 and DM2.
A number of diseases whose genetic bases are not yet known show anticipation, one of the hallmarks of the repeat expansion disorders. It may thus be that other diseases will be added to this group as their genetic basis becomes known. Available evidence suggests that some of these diseases may result from protein toxicity that is not associated with polyQ or RNA-mediated pathology that involves repeats other than r(CGG) or r(CUG).
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