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Human Molecular Genetics, 2003, Vol. 12, No. 1 41-50
© 2003 Oxford University Press

Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice

Randell T. Libby1, Darren G. Monckton5, Ying-Hui Fu6, Refugio A. Martinez1, John P. McAbney5, R. Lau7, David D. Einum8, K. Nichol7, Carol B. Ware2, Louis J. Ptacek8,9, Christopher E. Pearson7 and Albert R. La Spada1,3,4,*

1Department of Laboratory Medicine, 2Department of Comparative Medicine, 3Department of Medicine (Division of Medical Genetics) and 4Department of Neurology, University of Washington Medical Center, Seattle, WA, USA, 5Institute for Biomedical and Life Sciences, University of Glasgow, Glasgow, UK, 6Department of Human Genetics, University of California (San Francisco), San Francisco, CA, USA, 7Department of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada and 8Department of Human Genetics and 9Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT, USA

Received August 19, 2002; Accepted November 2, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the ataxin-7 gene. In humans, SCA7 is characterized by marked anticipation due to intergenerational repeat instability with a bias toward expansion, and is thus regarded as the most unstable of the polyglutamine diseases. To study the molecular basis of CAG/CTG repeat instability and its pathological significance, we generated lines of transgenic mice carrying either a SCA7 cDNA construct or a 13.5 kb SCA7 genomic fragment with 92 CAG repeats. While the cDNA transgenic mice showed little intergenerational repeat instability, the genomic fragment transgenic mice displayed marked intergenerational instability with an obvious expansion bias. We then went on to generate additional lines of genomic fragment transgenic mice, and observed that deletion of the 3' genomic region significantly stabilized intergenerational transmission of the SCA7 CAG92 repeat. These results suggest that cis-information present on the genomic fragment is driving the instability process. As the SCA7 genomic fragment contains a large number of replication-associated motifs, the presence of such sequence elements may make the SCA7 CAG repeat region more susceptible to instability. Small-pool and standard PCR analysis of tissues from genomic fragment mice revealed large repeat expansions in their brains and livers, but no such changes were found in any tissues from cDNA transgenic mice that have been shown to undergo neurodegeneration. As large somatic repeat expansions are absent from the brains of SCA7 cDNA mice, our results indicate that neurodegeneration can occur without marked somatic mosaicism, at least in these mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
At least 20 inherited disorders result from expansions of microsatellite or minisatellite repeats. Many of these diseases are caused by the expansion of a trinucleotide (1). When most CAG/CTG trinucleotide repeats expand into the disease-causing range, they show marked genetic instability. This genetic instability is characterized by key features including: (i) a tendency to expansion; (ii) an effect of parental sex upon the magnitude and direction of repeat instability and (iii) somatic mosaicism (i.e. marked variation in repeat lengths in different tissues of the body) (2). The phenomenon of genetic instability is interesting because it has clinical significance, as larger repeat expansions portend an earlier age of onset and worsening clinical severity for individuals affected with many of these diseases—a process called ‘anticipation’ (3). Anticipation may be accounted for by the tendency of trinucleotide repeats to expand as they are transmitted from one generation to the next.

Although the last decade yielded an explanation for the basis of anticipation, the mechanisms that underlie the repeat expansion process and its key features remain elusive. Introduction of repeat expansions into unicellular organisms suggested that replicative processes are at the core of repeat instability (4–7). Errors at replication forks have also been shown to produce CAG/CTG repeat expansions and deletions in a primate replication system (8). However, studies in cell culture and in mouse models suggest otherwise (9). Studies of SCAl and Huntington's disease (HD) transgenic mice bearing pathogenic CAG tracts indicate that expansion of trinucleotide repeats occurs in the absence of replication or meiotic recombination (10,11). In HD and myotonic dystrophy mouse models, mismatch repair function is necessary for marked somatic mosaicism (12,13). Consequently, our best models for trinucleotide repeat expansion posit the formation of aberrant DNA structures (14) that are a nidus for damage and thus an attempt at repair is made, but instead an alteration in triplet tract length in favor of expansion results (9). In addition to the proposed role of trans-acting factors in trinucleotide repeat instability, there is strong evidence supporting a role for cis-acting factors in this process (2,8,15). Perhaps, the best case for cis-control comes from the CAG polyglutamine repeat diseases where CAG tracts of identical length behave quite differently depending upon the locus at which they reside. For example, a CAG40 tract at the androgen receptor (AR) locus in spinal and bulbar muscular atrophy only changes length about one-quarter of the time that it is transmitted, and when it does so the length changes are small, ranging from -4 to +7 repeats (16). In SCA7, however, a CAG40 tract expands with most transmissions, and the alterations often involve jumps of >l0, sometimes >20, and occasionally >100 repeats (17).

Although the mechanism of somatic variability is unknown, recent analysis of this phenomenon in HD has yielded some provocative findings. Utilizing an ultra-sensitive method of repeat amplification and detection known as small-pool PCR (SP-PCR), one study of a HD CAG72-80 knock-in mouse model found that as many as 5% of the DNA molecules isolated from striatum measured >150 CAG repeats (18). While the implications of these findings remain speculative, one possible interpretation is that large expansions precede HD pathology and therefore play a role in disease pathogenesis. Further work on human HD tissues has confirmed that such enormously expanded repeats are present in a subset of cells from the striatum, the main focus of pathology in this disorder (P.F. Shelbourne, personal communication). Whether similar hyper-expansions of CAG tracts are present in the eight other polyglutamine repeat diseases remains to be determined.

To identify cis-acting factors responsible for CAG expansion, we studied instability at the SCA7 locus. Of all the CAG polyglutamine repeat diseases, SCA7 shows the most dramatic anticipation with many juvenile-onset cases and occasional infantile cases (19,20). To model instability, expanded CAG tracts from the human SCA7 locus were introduced into mice either on 13.5 kb genomic fragments or out of genomic context on ataxin-7 cDNAs. Comparison of the transmission of the SCA7 CAG repeats revealed that genomic context drives repeat instability with an obvious bias toward expansion, while SCA7 CAG repeats introduced on ataxin-7 cDNAs showed few length alterations. Deletion of the genomic region 3' to the SCA7 repeat stabilized the transmission of the CAG tract relative to that observed with longer genomic fragments in additional lines of mice, suggesting that cis-information conferring an instability potential to the SCA7 triplet resides within the 3' region. Using SP-PCR, we also found that large somatic expansions occurred in select tissues from the 13.5 kb genomic fragment mice, but similar expansions did not occur in tissues from cDNA mice. As the SCA7 cDNA transgenic mice show neurodegeneration (21,22), somatic expansions do not correlate with disease pathogenesis in this SCA7 mouse model.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Production and characterization of SCA7 transgenic mice
The ataxin-7 gene is ~140 kb, consists of 13 exons, and has its ATG initiation codon in exon 3 (23). Most SCA7 patients display CAG repeat expansions numbering from 38 to 130 triplets (24). To study repeat instability at the SCA7 locus, we first generated two types of transgenic constructs—a genomic fragment construct (RL-SCA7 series) and a cDNA construct (PrP-SCA7 series). The RL-SCA7 constructs consist of a 13.5 kb genomic fragment containing exons 3 and 4 of the ataxin-7 gene with ~5 kb of DNA 5' to the CAG repeat and ~8 kb of DNA 3' to the CAG repeat, including an entire CpG island (Fig. 1A and B). The PrP-SCA7 constructs contain the full-length ataxin-7 coding sequence cloned into the murine prion protein expression vector and incorporate only ~80 bp 5' and 210 bp 3' of genomic flanking DNA (Fig. 1C). Both control-length CAG10 or CAG24 and expanded CAG92 repeat versions of each construct were generated and then used to create transgenic mice, resulting in various founders, and ultimately eight independent transgenic lines. Breeding of these transgenic lines combined with Southern blot and quantitative PCR analysis allowed us to establish lines with a single transgene integration site and to determine the transgene copy number at each integration site (Table 1).



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  		Figure 1. Map of SCA7 gene and transgenic constructs. (A) The SCA7 gene consists of at least 13 exons with the CAG repeat located in exon 3. The 13.5 kb fragment used for the generation of the RL-SCA7 series of constructs is shown. (B) The RL-SCA7 genomic fragment transgenic constructs maintain the SCA7 CAG repeat in genomic context by subcloning the 13.5 kb fragment into pBluescript. Note that the CAG repeat is centrally located within this fragment. (C) The PrP-SCA7 cDNA transgenic constructs place the CAG repeat out of genomic context as only adjacent DNA sequence information from exon 3 is retained in this vector.

 

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  		Table 1. Genetic features of the PrP- and RL-SCA7 CAG repeat transgenic mice
 
Genomic context drives marked intergenerational repeat instability and expansion bias
We tracked the stability of the SCA7 CAG repeat as it was transmitted from parents to offspring through four generations of backcrossing onto the C57BL/6J background. To size repeat expansions, we performed PCR amplification on tail DNAs and resolved the resultant products by capillary gel electrophoresis (Fig. 2). Southern blot analysis and denaturing PAGE analysis confirmed the detection accuracy of this sizing (data not shown). PrP-SCA7 24R and RL-SCA7 10R mice with normal-length repeats showed perfect stability (Table 2). While the two PrP-SCA7 92R lines did reveal repeat length alterations upon intergenerational transmission, the frequency of such changes was small, ranging from 9.2 to 17.1% on a per transgene copy basis (Table 2). The RL-SCA7 92R genomic fragment lines, however, showed intergenerational repeat length changes in most instances, undergoing size shifts 65.3–83.0% of the time, on a per transgene basis. The frequency of alteration for the four RL-SCA7 lines was similar for all pairwise comparisons except one (see Table 2), and was significantly greater than the frequency of alteration for the PrP-SCA7 cDNA lines (P<0.0001) (Table 2).



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  		Figure 2. Representative examples of SCA7 CAG repeat sizing. (A) PCR amplification of the SCA7 CAG repeat region was performed for vector DNA (control) and three PrP-SCA7-92R transgenic mice (PrP-M1, PrP-M2 and PrP-M3). Genotyper scans of PCR products resolved on an ABI 310 Genetic Analyzer reveal tract size changes in the PrP-SCA7-92R mice. (B) PCR amplification of the SCA7 CAG repeat region was performed for vector DNA (control) and three RL-SCA7-92R transgenic mice (RL-M1, RL-M2 and RL-M3), Genotyper scans of PCR products resolved on an ABI310 Genetic Analyzer show detected repeat length changes.

 

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  		Table 2. Summary of the instability data for the PrP- and RL-SCA7 CAG repeat transgenic mice
 
In addition to displaying significantly higher frequencies of repeat length change, the RL-SCA7 mice also showed larger size changes and an obvious expansion bias in comparison to the PrP-SCA7 mice. For the PrP-SCA7 92R cDNA lines, length changes ranged from -4 to +5 repeats, with a mean length change of -0.1 repeat and a slight bias toward contraction (1.3:1) (Fig. 3). For the RL-SCA7 92R genomic lines, repeat length changes were much larger on average (P<0.001; t-test), ranging from -9 to +11 repeats with a mean repeat length change of +1.7 triplet per transmission (Fig. 3). Furthermore, there was considerable expansion bias upon transmission in genomic context (3.5:1 ratio), indicating that an expanded SCA7 CAG repeat is significantly more likely to further expand than to contract when in genomic context as compared to cDNA context (P<=0.005; Fisher's exact test). Despite the differences in repeat instability between the RL-genomic and PrP-cDNA transgenic lines, neither the RL-lines nor the PrP-lines showed a parent-of-origin effect upon the frequency of intergenerational tract length change (P=0.92; P=0.78) (Table 2). However, review of intergenerational instability for the PrP-cDNA lines revealed a profound effect of parent-of-origin upon the direction of repeat change for lines 6076 and 6529 (Fig. 3), with a strong bias towards contraction during maternal transmission (14:1) and an equally strong bias toward expansion during paternal transmission (11:1) (P<0.0001; Fisher's exact test). While an expansion bias characterized both maternal and paternal transmission for the RL-SCA7 genomic fragment lines, the ratio of expansion:contraction was more pronounced for paternal transmission (3.9:1) than for maternal transmission (2.2:1). This difference suggests a trend towards greater likelihood of expansion with paternal transmission of a SCA7 CAG92-99 repeat in genomic context, but was not significant (P=0.13; Fisher's exact test).



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  		Figure 3. Genomic fragment SCA7 transgenic lines show much more intergenerational repeat instability than the cDNA transgenic lines. Mutation spectra for intergenerational transmission of the SCA7 CAG repeat are shown for each transgenic line. Only a small fraction of the transgene transmissions show size alterations for the two PrP-SCA7 cDNA transgenic lines, 6076 and 6529. For the RL-SCA7 genomic fragment transgenic lines, 5735, 5736, 5746 and 5747, most of the transgene transmissions involve CAG repeat length changes. Note that the mutation spectra for the RL-SCA7 genomic fragment transgenic lines show a marked bias toward repeat expansion upon intergenerational transmission. Parental CAG repeat lengths for the PrP-SCA7 lines ranged from 91 to 94 triplets, while parental repeat lengths for the RL-SCA7 lines ranged from 92 to 99 triplets.

 
Genomic fragment mice lacking the 3' region display reduced repeat instability
To localize the DNA element(s) that drive the marked instability of the SCA7 CAG repeat, we initiated the production of various lines of transgenic mice with genomic fragments containing deletions. We began by designing a construct that lacks the majority of the flanking DNA 3' to the SCA7 CAG repeat (Fig. 4A). Microinjection of this so-called ‘alpha’ construct yielded three independent lines of {alpha}-SCA7 transgenic mice with single integration sites and single copy numbers based upon quantitative DNA PCR analysis (Table 3). We tracked the intergenerational transmission of the SCA7 CAG repeat through these three {alpha}-SCA7 92R transgenic lines and observed significant stabilization compared to the SCA7 13.5 kb genomic fragment lines (Table 3). Of the parent to offspring transmissions sized, we only observed repeat length alterations in ~47% of these transmissions (P<0.001 versus the RL-SCA7 92R lines; {chi}2). An even more dramatic difference was noted when we considered the range of repeat length changes occurring upon transmission of the SCA7 CAG repeat in the {alpha}-SCA7 92R transgenic lines. Repeat length changes ranged from just -3 to +2 triplets in the {alpha}-SCA7 92R lines, with expansions slightly outnumbering contractions to yield an expansion:contraction ratio of 1.3:1 (Fig. 4B). This range of repeat length changes, which corresponded to a mean repeat length change of just +0.2 triplet, indicates significant stability when compared with the range of repeat length changes observed in the RL-SCA7 92R lines (P<0.01; t-test). While significantly more stable than the RL-SCA7 genomic fragment lines, the frequency of instability in the {alpha}-SCA7 3' deletion lines is much greater than that observed in the PrP-SCA7 92R lines (P<0.001; chi-square), but the mean repeat length changes ({alpha}=+0.2 triplet; PrP=-0.1 triplet) and ranges ({alpha}=-3 to +2; PrP=-4 to +5) are similar between these two lines (P=0.17; t-test). We also considered the effect of parent-of-origin upon repeat instability in the {alpha}-SCA7 92R lines, and found that intergenerational instability did not differ significantly based upon the sex of the transmitting parent (P=0.42–1.0; overall P=0.23; {chi}2/Fisher's exact test). We did not observe an effect of parent-of-origin upon expansion-contraction tendency in the {alpha}-SCA7 92R lines (P=0.47; Fisher's exact test).



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  		Figure 4. Creation and analysis of genomic fragment 3' deletion transgenic lines. (A) A diagram of the {alpha}-SCA7 92R transgenic construct is shown. The {alpha} construct contains a large deletion of all flanking DNA beyond 245 bp 3' to the SCA7 CAG repeat (compared with the RL-SCA7 construct shown in Fig. 1B). (B) Deletion of 3' genomic flanking DNA stabilizes the SCA7 CAG repeat in the {alpha} transgenic lines. Mutation spectra for intergenerational transmission of the SCA7 CAG repeat are shown for three independent {alpha} transgenic lines, 629, 654 and 655. As shown here, alteration of CAG repeat length upon intergenerational transmission is much less frequent and is restricted to minor length changes in the {alpha}-SCA7 92R lines in comparison to the RL-SCA7 92R lines (see Fig. 3). Parental CAG repeat lengths for the {alpha}-SCA7 lines ranged from 92 to 104 triplets.

 

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  		Table 3. Results for the {alpha}-SCA7 CAG 92 repeat transgenic mice
 
Marked somatic mosaicism correlates with genomic context, not disease pathogenesis
For the RL-SCA7 92R mice, standard PCR amplification followed by denaturing PAGE analysis indicated that repeats larger than 160 CAGs were present in the brain and liver, but not in other tissues such as muscle or heart (Fig. 5A). That this length heterogeneity could be detected by standard PCR is an indication of considerable instability. SP-PCR was performed on RL-SCA7 92R tissues from mice ranging in age from 6 months to 15 months to confirm these changes, and revealed somatic mosaicism in a wide range of tissues (Fig. 5B; data not shown). Although CAG tracts ranging from 92 to ~120 triplets were present in kidney, retina, ovary, brain and liver from RL-SCA7 mice, large expansions, ranging from 120 to 250 CAGs, were confined primarily to liver and brain. Similar studies performed on PrP-SCA7 tissues from mice of similar ages indicated that the CAG92 repeat is remarkably stable somatically, as expansions greater than 100 CAG repeats were not detected by SP-PCR (Fig. 5B; data not shown). Absence of large repeat expansions in the brains and livers of the PrP-SCA7 92R mice stands in sharp contrast to the instability observed in identical tissues from RL-SCA7 92R mice.



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  		Figure 5. SCA7 genomic fragment transgenic lines show marked somatic instability, but somatic expansions are absent from SCA7 cDNA transgenic lines. (A) Standard PCR reactions performed on a variety of tissues from RL-genomic fragment transgenic mice (RL-M1 and RL-M2; 12 months and 15 months of age, respectively) reveal large somatic repeat expansions. The most notable somatic expansions are observed in the brain and liver samples from these mice. The numbers along the right side of the gel correspond to SCA7 CAG repeat numbers. Similar results were observed for additional RL-SCA7 92R mice ranging in age from 4 to 15 months (not shown). (B) Small-pool PCR reactions (using ~50 genome copies of DNA/reaction) were performed on a variety of tissues from PrP-SCA7 92R and RL-SCA7 92R mice ranging in age from 4 to 15 months for both the cDNA and genomic fragment lines. Representative SP-PCR results are shown for one PrP-SCA7 92R mouse (PrP-M1; 7 months of age) and two RL-SCA7 92R mice (RL-M1; RL-M2). While the SCA7 CAG repeat is remarkably stable in size in tissues from PrP-M1, somatic mosaicism is quite apparent in tissues from RL-M1 and RL-M2. Note that large somatic expansions, numbering from 120 to 250 CAG repeats are discernible in the brain and liver of the RL-SCA7 mice, with considerable expansions of 110–180 CAG repeats present in kidney, retina and ovary in RL-M1. The numbers along the right side of the gel correspond to SCA7 CAG repeat numbers.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although significant progress has been made in understanding the mechanism of polyglutamine neurodegeneration over the past decade, the pathways and molecular players orchestrating CAG/CTG repeat instability have remained elusive. Furthermore, the relevance of tissue-specific repeat expansions to polyglutamine disease pathogenesis has also recently come into question. In this study, we introduced different versions of an expanded CAG repeat into the germline of mice as a way to study the basis of repeat instability. Only limited intergenerational instability with a slight tendency to repeat contraction was observed in two independent lines of PrP-SCA7 92R transgenic mice carrying either one or two cDNA transgene copies. In contrast, marked intergenerational repeat instability with a strong expansion bias was observed in four independent lines of RL-SCA7-92R transgenic mice carrying either one or two genomic transgene copies. We conclude that such differences in repeat instability between the cDNA and genomic fragment transgenic lines reflect a requirement of cis-elements specific to the SCA7 genomic context. While the importance of genomic context in promoting instability in transgenic mice has been suggested (25–27), the present study is the first and only direct comparison of a cDNA construct and a genomic fragment construct carrying identically sized CAG/CTG repeat expansions from the same locus. Furthermore, we eliminated copy number variation and genetic strain background as confounding factors by backcrossing all lines onto the C57BL/6J strain. For these reasons, we believe that the present study validates the notion that a wider genomic context drives repeat instability in transgenic mouse models of CAG/CTG expansion tracts.

As a follow-up to the finding of the dependence of SCA7 CAG repeat instability upon genomic context, we generated deletion constructs to hone in upon the genomic region(s) responsible for the marked instability at the SCA7 locus. Our first set of such deletion constructs, which we designated as the {alpha}-SCA7 92R series, lack most of the 8 kb of 3' DNA flanking the SCA7 CAG repeat that is present in the RL-SCA7 92R constructs. Generation of three independent lines of {alpha}-SCA7 92R mice allowed us to determine whether or not sequence information 245 bp beyond the 3' end of the SCA7 CAG repeat is required for the marked repeat instability observed in the RL-SCA7 92R genomic fragment mice. Both the significantly decreased frequency of repeat length changes and the much narrower range of repeat size changes observed upon transmission of the SCA7 CAG repeat in the {alpha}-SCA7 92R lines indicate that cis-information required for marked intergenerational instability may indeed reside within the 3' genomic region. Comparison of intergenerational repeat instability between the {alpha}-SCA7 3' deletion mice and the PrP-SCA7 cDNA mice, however, did reveal significantly more frequent repeat-length alteration in the {alpha}-SCA7 mice, suggesting that some elements retained within the 5' flanking genomic sequence are capable of mediating a degree of intergenerational instability. While previous studies of repeat instability in CAG/CTG transgenic mice have implicated repeat length, sex of the transmitting parent, and sex of the recipient offspring as factors regulating repeat instability (11,28,29), our results suggest that cis-information plays a key role in the repeat instability process.

Of all the polyglutamine diseases, SCA7 shows the greatest degree of anticipation as it exhibits significantly more instability and larger repeat expansions than the other eight CAG repeat diseases, despite showing considerable overlap in pathological size ranges (17,30). We hypothesize that the increased instability at the SCA7 locus reflects the presence of flanking sequences that somehow drive this process. In a previous study, one of us demonstrated that the SCA7 CAG repeat is in a region of high percentage GC content and that high percentage GC content is associated with greater levels of instability (15). As the RL-genomic mice show significantly greater repeat instability than the PrP-cDNA mice, it is interesting to note that the high percentage GC content of the SCA7 genomic region appears to correlate with a high density of replication-associated motifs (Fig. 6). Many of these motifs correspond to sequence elements shown to be present at putative replication origins (31,32), and all are contained within introns 3 or 4 of the SCA7 genomic fragment and thus not present in the PrP-cDNA construct fragment. Recent evidence suggests that CAG/CTG instability can occur by mechanisms including errors at primate replication forks (8). Alternatively, the presence of numerous replication-associated motifs is known to correlate with altered DNA helical instability (31–33), suggesting that the SCA7 repeat region may be susceptible to changes in DNA secondary structure. In this regard, it is interesting to note that the DNA region 3' to the SCA7 CAG repeat contains the vast majority of these replication-associated motifs and appears to be required for marked intergenerational repeat instability in transgenic mice. If the adoption of alternative DNA secondary structures is a prerequisite for DNA damage such as nicking and, presumably, for subsequent DNA repair, it could explain the marked instability at the SCA7 locus. The goal of future studies will be to determine whether any such motifs contained in the 13.5 kb SCA7 genomic fragment are required for promoting repeat instability.



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  		Figure 6. Frequency of modular sequence elements associated with recombination and replication in the CAG repeat regions of the human SCA7 genes. Exons are indicated as open boxes underneath the genomic fragment maps, and the locations of the various identified elements (divided in some cases based on the degree of homology) are shown as hash marks. That the vicinity of the SCA7 CAG repeat is uncharacteristically rich in replication-origin associated sequences is supported by the results of an identical screen of a 13.5 kb genomic fragment from the human AR locus that yielded only six near perfect matches for replication-associated motifs and no origin-like sequences. Abbreviations are as follows: SAR, scaffold attachment region; ARS, autonomously replicating sequence; Topo II, topoisomerase LI binding site; Pur, pur protein binding site; Hu Rep Origin, human replication origin-like sequence.

 
To determine if repeat expansion within affected tissues takes place, we studied somatic mosaicism in our SCA7 transgenic mice. Repeats were perfectly stable in brains of the PrP-SCA7 92R mice, but large repeat expansions were present in brains and livers of the RL-SCA7 92R mice. The detection of large somatic repeat changes in the RL-SCA7 92R mice indicated that proper genomic context may be necessary for marked somatic instability with a 92 CAG repeat from the SCA7 locus. Since RL-SCA7 mice are phenotypically normal, our studies demonstrate that large somatic expansions are not merely the by-product of cellular dysfunction or degeneration. Another significant implication of this work is that large somatic expansions need not occur in the brains of PrP-SCA7 transgenic mice to produce dysfunction and neurodegeneration. Given these results, a potential contribution of somatic instability to the PrP-SCA7 neurological phenotype cannot be determined. In the case of HD, evaluation of a knock-in mouse model and human patient material has yielded evidence for the presence of large somatic expansions in tissues destined to degenerate (18) (P. Shelbourne, personal communication). Whether such large somatic expansions are present in SCA7 or in the other seven non-HD polyglutamine repeat diseases remains to be determined. Thus, while the absence of large somatic expansions in the PrP-SCA7 cDNA transgenic mice suggests that repeat instability is not absolutely required for disease pathogenesis, our results do not exclude a role for somatic mosaicism in SCA7 disease pathogenesis. Indeed, if the RL-SCA7 mice are recapitulating the mutational pathway in man, then the pattern of somatic mosaicism observed in the RL-SCA7 mice is consistent with a possible role for large repeat expansions in the SCA7 disease process. Only with the development of additional knock-in, BAC and YAC transgenic models of the polyglutamine repeat diseases and with careful analysis of human patient material will the relationship between somatic instability and disease pathogenesis be clarified. In the interim, the PrP-SCA7 mice should serve as an excellent model system to evaluate polyglutamine neurotoxicity in the absence of the confounding effects of somatic mosaicism.

The RL-SCA7 92R and PrP-SCA7 92R transgenic lines differ not only in the sequence context for the CAG repeat but also in the ability of the repeat to be expressed. As we have reported, the PrP-SCA7 92R mice develop a cone–rod dystrophy type of retinal degeneration due to expression of polyglutamine-expanded ataxin-7 in the neural retina (22). The PrP-SCA7 92R mice also show brainstem neurodegeneration and widespread central nervous system dysfunction (21). In the case of the RL-SCA7 92R mice, no deleterious phenotype is ever observed and RT-PCR studies fail to detect expression from the short open reading frame contained in the two coding exons (data not shown). These results indicate that the DNA structural changes accompanying generation of a transcriptional activation complex are not sufficient to mediate high levels of intergenerational instability or somatic mosaicism in the PrP-SCA7 cDNA mice, and are not required for intergenerational instability or somatic mosaicism in the RL-SCA7 genomic fragment mice. Thus, our studies of repeat instability in the SCA7 transgenic mice disconnect gene transcription from marked trinucleotide repeat instability, both in the germline and in somatic tissues.

The purpose of this work was to use the mouse as a model organism for the study of the repeat instability mechanism and its pathological significance. While our SCA7 genomic fragment transgenic mice did not exhibit large intergenerational repeat expansions of the size seen in humans, the marked repeat instability and significant expansion bias observed in these mice are reminiscent of the behavior of this repeat at its human locus. Importantly, independent lines of SCA7 cDNA transgenic mice carrying 92 CAG repeat expansions showed little instability, either somatically or intergenerationally, while multiple lines of 13.5 kb SCA7 genomic fragment mice of comparable genetic strain background carrying identically sized CAG repeat expansions showed marked repeat instability, both somatically and intergenerationally. The presence of numerous replication-associated motifs within the SCA7 genomic fragment construct offers a number of possible targets that might account for this difference in instability potential. Based upon our finding of significant intergenerational stability in SCA7 genomic fragment mice with deletion of the 3' genomic region, it appears that the search for any putative DNA instability element(s) should be focused upon the region of 3' DNA flanking this CAG repeat. The goal of future studies will thus be the identification of the precise cis-acting elements and trans-acting factors that underlie CAG/CTG repeat instability at the SCA7 locus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Transgenic constructs and production of transgenic mice
PCR primers spanning the 5' UTR (hSCA7-5F: 5'-agagaaacttggcgacctc-3'; hSCA7-5R: 5'-tcctcatccacctctcacc-3') and 3' UTR (hSCA73F: 5'-gccgggctgttgttttaacg; hSCA73R: 5'-cgtggaattgacctctggtc-3') of the ataxin-7 gene were used to screen a human YAC library. The identified YAC (762e11) was subcloned into SuperCos I vectors (Stratagene) that were screened for the CAG repeat, thereby yielding Cos SCA7-7. The RL-SCA7 10R transgenic construct was made by subcloning a 13.5 kb Sal I-Spe I genomic fragment from Cos SCA7-7 into pBluescript KS+ (Stratagene). The RL-SCA7 92R transgenic construct was made by subcloning an Aat II-Nar I fragment containing 92 repeats from a SCA7 cDNA (22). RL-SCA7 transgenic founders were generated by microinjection of purified Sal I-Spe I DNA fragments, and founders identified by PCR amplification of the CAG repeat (see below). The PrP series of transgenic constructs and mice have been described (22). The Alpha construct was generated by digesting the RL-SCA7 92R construct with Nru I and Spe I, thereby releasing an ~8.3 kb fragment. The Nru I and Spe I sites were then filled in with Klenow fragment of DNA polymerase, and the vector was ligated overnight prior to transformation. Embryos for pronuclear microinjection were the result of matings between C57BL/6J males and C57BL/6JxC3H/HeJ Fl females. Founders were backcrossed onto the C57BL/6J strain background, as were all subsequent transgenic progeny.

PCR analysis
For intergenerational repeat instability, DNA was isolated from tail biopsies by standard protocol (34). The SCA7 CAG repeat was PCR-amplified using primers 5' FAM-2 tcggagcgggccgcggatgac-3' and 5'-cacgactgtcccagcatcactt-3' in 20 µl reactions containing 2.0 µl 10x PE buffer, 1.0 µl 25 mM MgCl2, 0.15 µl 10 µM of each primer, 6.0 µl betaine, 0.4 µl 10 mM dNTPs, 1.5 U AmpliTaq (ABI), and 20 ng DNA. After a pre-denaturation step at 94°Cx5 mm, 32 cycles of 94°x45s, 60°Cx1 min, and 72°Cx1 min were performed with a final extension at 72°Cx10 min. PCR-amplified DNA was sized on an ABI 310 Genetic Analyzer relative to the ROXl000 DNA size standard using GeneScanTM 2.0.2 and Genotyper software (ABI). For somatic mosaicism analysis, DNA was isolated from different tissues and analyzed as above or by SP-PCR as previously described, using primers SCA7-A and -BR (RL-mice) or primers SCA7-BR and -E (5'-gaattcgccaccatgtcggag-3') (PrP-mice) (20,35).

Quantitative real-time PCR
SCA7 copy number was determined using the TaqMan approach on an ABI7700 Sequence Detection System. Primers and probe (available upon request) were added at 300 nm to 40 µl reactions. Fifty cycles of 95°Cx15 s followed by 60°Cx1 min were run, with reaction concentrations as specified (ABI). All amplifications were run in triplicate and a standard curve generated, based on control copy number reactions. Copy number was then determined by comparison of standard curve ct values with the ct values obtained for transgenic samples.

Southern blot analysis
A 1.7 kb EcoRI SCA7 genomic fragment (clone kindly provided by S. Richards and B. Roa) was radiolabeled and hybridized to nylon filters onto which EcoRI-digested mouse genomic DNAs had been transferred (36).

Sequence analysis
Comparable segments of the SCA7 and AR genes were screened for motifs associated with recombination and replication (31–33,37). Pairwise sequence comparisons were carried out using LALIGN (www.ch.embnet.org/software/LALIGN_form.html).

Statistical analysis
Chi-square and t-test analyses were performed using Microsoft Excel, 98. The Fisher's exact test was run using the Langsrud website (www.matforsk.no/ola/fisher.htm).


   ACKNOWLEDGEMENTS
 
The authors wish to thank P. Huynh for technical assistance, and P. Shelbourne for her permission to include unpublished material. This work was supported by NIH grant GM59356 (A.R.L.), by funding from the Canadian Institutes of Health Research (C.E.P.), and by a generous gift from a Utah SCA7 family (Y.-H.F.). A.R.L. is a Paul Beeson American Foundation for Aging Research Scholar, D.G.M. is a Lister Institute Research Fellow, and C.E.P. is a Canadian Institutes of Health Research Scholar and a Canadian Genetic Disease Network Scholar.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Laboratory Medicine, University of Washington Medical Center, Box 357110, Room NW 120, Seattle, WA 98195-7110, USA. Tel: +1 2065982138; Fax: +1 2065986189; Email: laspada{at}u.washington.edu Back


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