Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia (original) (raw)

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Published:

01 January 2004

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Laura M. Pollard, Rajesh Sharma, Mariluz Gómez, Sonali Shah, Martin B. Delatycki, Luigi Pianese, Antonella Monticelli, Bronya J.B. Keats, Sanjay I. Bidichandani, Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia, Nucleic Acids Research, Volume 32, Issue 19, 1 October 2004, Pages 5962–5971, https://doi.org/10.1093/nar/gkh933
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Abstract

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.

Received July 22, 2004; Revised and Accepted October 22, 2004

INTRODUCTION

Friedreich ataxia, the most prevalent inherited ataxia, is caused by abnormal expansion of a GAA triplet repeat sequence in intron 1 of the FRDA gene (1,2) (http://www.geneclinics.org/). This sequence is both polymorphic and genetically unstable (1,3,4). Normal alleles, which contain <32 triplets, are classified as being either short normal (SN; <12 triplets) or long normal (LN; ≥12 triplets). Disease-causing expanded (E) alleles, containing 66–1700 triplets, interfere with gene transcription at the _FRDA_ locus, thereby resulting in frataxin deficiency and Friedreich ataxia (5–9). SN and LN alleles are stably transmitted from parent to offspring (3,4). However, E alleles display intergenerational instability, usually contracting by 20–30% upon paternal transmission, and showing equal tendencies for expansion and contraction during maternal transmission (10,11). Rare, intermediate-sized alleles, containing 33–65 uninterrupted GAA triplets, are termed premutation (PM) alleles because they may undergo hyperexpansion upon intergenerational transmission, expanding by >5- to 10-fold to generate E alleles in offspring [(3,4,12,13); unpublished data]. PM alleles and those at the long end of the LN spectrum may be interrupted by (GAGGAA)n hexanucleotides, which are believed to result in their stabilization during germline transmission (3). By analyzing individual somatic genomes from peripheral blood leukocytes using small-pool PCR (SP-PCR), we previously showed that E alleles are extremely unstable in vivo and show a marked predilection for large contractions (14).

Unstable triplet repeats are known to cause at least 15 inherited diseases (15,16). So far, only three of the ten possible triplet repeat motifs are known to be associated with disease: (CGG•CCG)n, (CTG•CAG)n, and (GAA•TTC)n. Instability of (CGG•CCG)n and (CTG•CAG)n repeat sequences in bacteria and yeast is dependent upon both repeat length and the orientation of the repeat relative to the origin of replication (17–26). For example, the (CTG•CAG)n repeat tract is more unstable, with a significant tendency for contractions, when CTG is the template for lagging strand synthesis. Either strand of the (CGG•CCG)n or (CTG•CAG)n repeat sequence (i.e. the CNG triplet motif) has a tendency to form hairpin structures. The relative thermodynamic stability of the hairpin formed by single-stranded CTG versus CAG on the lagging strand template is thought to be the basis for the contraction bias in both bacterial and yeast systems (17).

Recent evidence suggests the possibility that GAA and TTC single-stranded sequences may form hairpin structures (27), which is a well-known property of both the CNG triplet repeats (28). However, the polypurine•polypyrimidine (GAA•TTC)n repeat differs from the two unstable CNG triplet repeats in its ability to adopt triplex structures in supercoiled templates (5,7,29–32). Although a triplex structure formed by the (GAA•TTC)n repeat is believed to be the molecular basis for the transcriptional interference afforded by expanded FRDA alleles (5–7,9,30,33), its role in mediating genetic instability is less clear. We proposed that errors during lagging strand synthesis could be the basis for the genetic instability of the (GAA•TTC)n repeat based on our previous observation of large, spontaneous GAA contractions in somatic cells in vivo (14), the known disparity in the relative affinities of replication protein A (RPA) for polypurine versus polypyrimidine sequences (34) and the propensity of single-stranded (GAA)n sequence to adopt altered structural conformations (35,36). Furthermore, Ohshima et al. (6) had previously reported orientation-dependent instability of (GAA•TTC)n repeats, noting increased smearing of their cloned inserts after restriction digest, indicative of a contraction bias in plasmids when GAA was the template for lagging strand synthesis.

Therefore, in this study we conducted a more detailed investigation of the effect of orientation of DNA replication on the instability of repeats, cloned from human carriers of several FRDA alleles, in plasmids propagated in Escherichia coli. We also evaluated the instability of similar repeat lengths in peripheral blood leukocytes from human donors using SP-PCR. Here, we show that while small slippage-type events occurred in both orientations, large contractions of (GAA•TTC)n sequences occurred primarily when GAA was the template for lagging strand synthesis. DNA replication was significantly impeded in this orientation, and the incremental accumulation of large contractions was coincident with the phase of maximal plasmid replication. We also observed the same spectrum of mutations in peripheral leukocytes in vivo in human subjects carrying FRDA alleles of similar length, including complete reversions to the normal/premutation length. During the preparation of this manuscript, similar findings were reported using (GAA•TTC)n containing plasmids in yeast (37). This indicates that the mechanism of the strand-specific differential instability of (GAA•TTC)n repeats appears to be conserved among prokaryotes and eukaryotes, and the contraction bias is seen not only in prokaryotes and simple eukaryotes, but in human somatic cells as well. The threshold length for the initiation of somatic instability in vivo coincided with the length of a eukaryotic Okazaki fragment (38,39). (GAGGAA)n hexanucleotide interruptions stabilized the (GAA•TTC)n sequence in E.coli and human somatic cells, constituting the first direct evidence for a stabilizing role of interruptions in the context of the human genome in vivo.

MATERIALS AND METHODS

Plasmid construction

Genomic DNA from whole blood of human subjects was used to amplify 9, 21, 44, 81, 107, 119 and 163 pure (GAA•TTC)n repeats and 114 (GAA•TTC)n repeats containing a (GAGGAA)n hexanucleotide repeat interruption in intron 1 of the FRDA gene, using the following primers (40): GAA-104F (5′-GGCTTAAACTTCCCACACGTGTT-3′) and GAA-629R (5′-AGGACCATCATGGCCACACTT-3′), followed by nested PCR using one of the following primer pairs: gaapst1-F (5′-GCTCCGCTGCAGCCCAGTATCTACTAAAAAATAC-3′) and gaaxba1-R (5′-GATGCGTCTAGACGCGCGACACCACGCCCGGCTAAC-3′), or ttcpst1-F (5′-GCTCCGCTGCAGCGCGCGACACCACGCCCGGCTAAC-3′) and ttcxba1-R (5′-GATGCGTCTAGACCCAGTATCTACTAAAAAATAC-3′).

The PCR products were digested with PstI and XbaI, which recognize sequences located at the 5′ end of the forward and reverse primers, respectively. The fragments containing (GAA•TTC)n repeats along with minimal flanking sequence (38 bp 5′ and 35 bp 3′ to the repeat) were ligated into the PstI and XbaI sites of pUC19. The pUC19 vector used contains the ColE1 origin and a portion of the ROP gene making it a low copy number plasmid. The primer pairs determined the orientation of the repeat tract: ‘GAA’ orientation (gaapst1-F/gaaxba1-R) or ‘TTC’ orientation (ttcpst1-F/ttcxba1-R) (Figure 1). The following recombinant plasmids were selected for mutation analysis: 9, 21, 48, 82, 105 and 111 repeats in the ‘GAA’ orientation, and 8, 21, 48, 79, 105 and 108 repeats in the ‘TTC’ orientation. Repeat lengths and orientation were determined by sequencing. The constructs containing (GAA)9, (GAA)48, (GAA)105, (GAA)111, (TTC)8, (TTC)48, (TTC)79, (TTC)105 and (TTC)108 were pure (GAA•TTC)n repeats, and the other constructs contained either a single non-GAA interruption within the repeat tract as follows: (GAA)21 = (GAA)17(A)(GAA)4; (GAA)82 = (GAA)70(AA)(GAA)11; (TTC)21 = (TTC)4(TT)(TTC)17 or a (GAGGAA)5 hexanucleotide interruption along with additional non-GAA sequence as follows: (GAA)101* = (GAA)60GA(GAA)2GAGGA(GAA)18(GAGGAA)5(GAA)8; (TTC)95* = (TTC)8(TTCCTC)5(TTC)18TCCTC(TTC)2TC(TTC)54. A control, non-repeat containing plasmid with a 289 bp insert of random sequence (51% G/C content) was created by amplifying exon 5 of the APTX (aprataxin) gene using primers: aptx5pst1-F (5′-GCTCCGCTGCAGGTCTGTTTCCTTCTCTTGT-3′) and aptx5xba1-R (5′-GATGCGTCTAGAGGAGCCAGCAGCACTACC-3′), which was similarly digested and inserted into the PstI and XbaI sites in pUC19.

Figure 1.

(GAA•TTC)n constructs used to analyze the effect of differential orientation of replication on repeat instability. Several lengths of the (GAA•TTC)n sequence were cloned into the PstI/XbaI sites of pUC19 in both orientations with respect to the unidirectional ColE1 origin of replication (arrow). Repeat-containing constructs are depicted in the ‘GAA’ or ‘TTC’ orientations, with repeat lengths of n = 9, 21, 48, 82, 105 and 111 in the ‘GAA’ orientation, and n = 8, 21, 48, 79, 105 and 108 in the ‘TTC’ orientation. Additionally, two interrupted tracts of 101* and 95* repeats were cloned in the ‘GAA’ and ‘TTC’ orientation, respectively. The black boxes flanking the repeat represent minimal flanking sequence from intron 1 of the FRDA gene.

Analysis of (GAA•TTC)n repeat instability

Individual bacterial (DH5α) colonies obtained by plating glycerol stocks of sequence verified plasmids were grown in liquid culture (Luria–Bertani + 100 μg/ml ampicillin) at 30°C for 12–24 h. PCR of individual colonies was performed to confirm that the inserts were full-length repeat tracts immediately prior to initiation of cultures. Colonies representing each repeat length and orientation of replication were cultured in triplicate, so that at each hour (between 12–24 h) three separate 5 ml cultures could be harvested for each construct. Detailed studies were carried out for GAA-82, TTC-79 and a random sequence insert, in triplicate. Each culture was treated as follows: 1 ml was used to estimate the OD600 as a measure of cell density (to plot bacterial growth curves), 1 ml was used to set up a glycerol stock (for mutation studies, see below) and the rest was used for plasmid DNA isolation (to measure the amount of plasmid replication). Plasmid DNA was applied to a Zeta-Probe® membrane using a Bio-Dot® microfiltration apparatus (BioRad), probed with γ-ATP32-labeled pUC19-R oligonucleotide and quantified by densitometry. Bacterial growth curves and plasmid replication results were used to determine the time points for detailed mutation analysis: 12, 16, 20 h (log phase), and 24 h (stationary phase) of culture. We specifically avoided multiple sub-culturing of E.coli and only analyzed the mutational profile over a single log phase, which resulted in low mutation frequencies (>80% of final inserts were full-length), thus minimizing the bias for contractions and multiple mutations involving the same repeat tract.

Previous reports indicate that transformation of triplet repeat-containing plasmids per se increases the instability of the repeat tract (41). Therefore, glycerol stocks for GAA-82 and TTC-79 at 12, 16, 20 and 24 h time points were plated and incubated at 37°C for 16 h. PCR of individual colonies was performed to assess (GAA•TTC)n repeat instability using primers: GS-F = (5′-CCCAGTATCTACTAAAAAATAC-3′) and GS-R (5′-ACACCACGCCCGGCTAACTTTTC-3′). Relative sizes of PCR products were determined by electrophoresis on 3% agarose gels, coupled with direct sequencing of selected products. Approximately 100 colonies were analyzed for each orientation and time point, in triplicate, and instability was defined as the percentage of the total number of PCR products amplified whose length was altered after replication in E.coli. Large contractions were defined as products that lost >50% of their initial repeat length.

To compare E.coli growth curves and instability of pure versus interrupted (GAA•TTC)n repeat tracts, 5 ml cultures were inoculated with colonies containing plasmids with either the pure or interrupted repeat tract inserted in both orientations. Cultures were grown at 30°C for 10–24 h, in triplicate, such that three cultures for each construct were harvested every hour. Growth curves were generated as described above. Repeat tract instability was analyzed after 22 h of growth, as described previously. Approximately 75 colonies were analyzed for each of the four constructs, in triplicate.

SP-PCR analysis

The SP-PCR analysis was carried out as described previously (14,42). Briefly, serial dilutions of human genomic DNA, ranging from 6–600 pg, were prepared in siliconized microfuge tubes. PCR was performed using GAA-104F and GAA-629R, which allowed accurate sizing of alleles used in the present study. PCR products were resolved by electrophoresis on 1–2% agarose gels (2% gels were used for the calculation of threshold length for the initiation of instability), and bands detected by Southern blotting using an end-labeled (TTC)9 oligonucleotide probe. The calculation of the average number of individual FRDA molecules per reaction (the ‘gene equivalent’) was performed by Poisson analysis as described previously (42,43). The average quantity of genomic DNA required for the amplification of one FRDA molecule was 13.4 pg (95% CI 10.6–16.1 pg). For each genomic DNA sample, multiple reactions were performed using ‘small pools’ of 2.5–25 individual FRDA molecules per reaction to detect mutations. Mutation loads were calculated as the proportion of molecules that differed by >5% in size from the constitutional (most common) allele determined by direct sequencing.

Statistical methods

Comparison of medians was carried out using the Mann–Whitney U test, means were compared using the _t_-test, and frequencies were compared by χ2 analysis.

RESULTS

Large contractions of (GAA•TTC)n occur when ‘GAA’ is the template for lagging strand synthesis

(GAA•TTC)n repeat tracts (n = 9, 21, 48, 80 and 105 triplets) were cloned in both orientations relative to the ColE1 origin of replication, such that either GAA or TTC would serve as the template for lagging strand synthesis (Figure 1). Replication of these cloned (GAA•TTC)n triplet repeats, which were propagated in E.coli (DH5α), displayed length-dependent instability. Whereas repeat tracts containing ≤21 triplets were completely stable, repeat tracts containing 48 and ∼80 triplets were moderately unstable and the construct containing ≥100 triplets in the GAA orientation was highly unstable (data not shown). We selected the moderately unstable repeat tracts containing ∼80 triplets to perform a detailed analysis of the effects of differential orientation of replication on GAA triplet repeat instability, which was accomplished by analyzing multiple individual replication events in bacterial colonies. The (GAA•TTC)n sequence was found to be significantly more unstable when GAA was the template for lagging strand synthesis (‘GAA’ orientation; GAA-82) compared with TTC (‘TTC’ orientation; TTC-79) (Figure 2A). The same orientation-dependent instability was also noted when (GAA•TTC)n repeats were cloned in plasmids pBluescript II (Stratagene), pCR2.1 and pCR3.1 (Invitrogen) and replicated in DH5α and TOP10 (Invitrogen) strains of E.coli (data not shown).

Figure 2.

Slowed replication and large contractions of the (GAA•TTC)n repeat when GAA is the template for lagging strand synthesis. Closed diamond = GAA-82, closed square = TTC-79, closed triangle = random sequence control and all error bars reflect ± SD (A) Representative PCR products generated from colonies obtained by plating the glycerol stock of a single culture (16 h) are shown. The repeat tract was significantly more unstable and prone to contractions when GAA was the template for lagging strand synthesis. Arrows indicating the position of GAA-40 and TTC-40 represent the cutoff (∼50%) used for defining small versus large contractions. (B) Slower growth of E.coli and significantly blunted log phase when GAA was the template for lagging strand synthesis compared with TTC or random sequence control. (C) Slower plasmid DNA replication when GAA was the template for lagging strand synthesis compared to TTC or random sequence control, as determined by dot blot analysis. RDU, relative densitometric units. (D) Percentage of colonies containing (GAA•TTC)n repeats of altered length (% instability) after 12, 16, 20 and 24 h of culture indicates that there was a significant increase in instability over time for GAA-82 versus TTC-79 (P = 0.01 at 20 and 24 h). (E) Large contractions (>50% loss of initial repeat length) were significantly more frequent with GAA-82 versus TTC-79 (P < 0.001). The median length of contraction products (indicated by horizontal lines) was significantly shorter for GAA-82 (25.5 repeats) than for TTC-79 (49.5 repeats) (P < 0.001). Only contraction events are shown in the graph, with the magnitude of change (in triplets) plotted on the y-axis. (F) Large contractions accumulated throughout the log phase when GAA was the template for lagging strand synthesis.

To further examine the role of replication in GAA triplet repeat instability, we analyzed the mutational frequencies and spectra during the entire period of active replication of plasmids in E.coli. Throughout the log phase of E.coli growth, both bacterial growth and plasmid DNA replication were significantly slower when GAA was the template for lagging strand synthesis (Figure 2B and C). A random sequence insert of the same size showed the same profile as when TTC was the lagging strand template (Figure 2B and C). Even when cultures were grown for 40 h, well into the stationary phase, bacterial density was significantly lower for GAA-82 than for either TTC-79 or the random sequence control (data not shown). These data indicate that DNA replication is significantly slower when GAA is the template for lagging strand synthesis.

Repeat instability was analyzed for GAA-82 and TTC-79 after 12, 16, 20 (log phase), and 24 h (stationary phase) of culture. Instability was significantly greater in the ‘GAA’ orientation, and it increased throughout the exponential phase of plasmid replication (Figure 2D). Contractions were much more prevalent than expansions in both orientations. However, analysis of all contraction events from 12–24 h revealed that large contractions, involving >50% of the original length, were observed primarily when GAA was the template for lagging strand synthesis (Figure 2A and E). Large contractions in the ‘TTC’ orientation were significantly less frequent (P < 0.001), and of lesser magnitude [median size of contractions = 25.5 versus 49.5 triplets (P < 0.001)]. Furthermore, despite the relatively blunted exponential replication phase, there was an incremental accumulation of large contractions throughout the log phase in the GAA orientation (Figure 2F), and indeed, most of the observed instability was due to these large contractions (Figure 2D and F).

We did not observe large expansions in either orientation of replication. Small contractions and expansions (involving <10% of the original tract length) were equally frequent in the GAA or TTC orientations (P = 0.41 and P = 0.37, respectively). Large contractions were far more prevalent than small contractions only in the ‘GAA’ orientation (P < 0.001) (Figure 2E). In summary, we noted large contractions predominantly when GAA is the lagging strand template, and this was coincident with plasmid DNA replication.

Similar mutational spectrum is observed at the FRDA locus in human somatic cells in vivo

To test the physiological relevance of our observations, we performed SP-PCR analysis of individual FRDA molecules containing repeat lengths similar to those tested in E.coli, derived from genomic DNA of peripheral blood leukocytes of human subjects. We have previously optimized this technique for the analysis of GAA triplet repeat instability at the FRDA locus (14). Analysis of 2520 individual FRDA molecules from four heterozygous carriers of constitutional alleles with 78 (n = 524 molecules), 81 (n = 700 molecules), 91 (n = 328 molecules) and 105 (n = 968 molecules) uninterrupted triplet repeats revealed the same spectrum of mutations observed in E.coli: small expansions and contractions, large contractions and a paucity of large expansions or intermediate-sized contractions (Figure 3A and B). Of the 2520 molecules analyzed, we observed 283 variant bands (mutation load = 11.2%), with a significant contraction bias [55 (2.2%) expansions versus 228 (9%) contractions, _P_ < 0.001; Figure 3B]. In contrast to replication in E.coli, somatic instability in leukocytes resulted in far more small versus large contractions. However, the extent of the large contractions was similar in the two systems, with most large contractions resulting in almost complete reversion to the normal size, and very few intermediate-sized contractions (Figure 3B). It should be noted that the frequency of large contractions is most likely underestimated in our SP-PCR assay since all four carriers used in this study also have a normal allele, which makes it nearly impossible to detect ‘complete’ reversion events of individual FRDA molecules (Figure 3A).

Figure 3.

SP-PCR analysis of (GAA•TTC)n alleles with 78–105 uninterrupted repeats shows instability and a contraction bias in somatic cells in vivo. (A) A representative Southern blot showing multiple ‘small pools’ of FRDA molecules from human genomic DNA containing 9 and 78 GAA triplet repeats. Somatic instability in vivo comprised frequent small contractions/expansions and some large contractions into the normal, non-disease size range. (B) Summary of mutations detected following SP-PCR analysis of 2520 individual FRDA molecules containing (GAA•TTC)n with n = 78, 81, 91, or 105 uninterrupted repeats. The x-axis represents the magnitude of change (%) from the constitutional (most common) GAA triplet repeat length, determined by sequencing, with negative and positive readings indicating contractions and expansions, respectively. Non-mutant bands are not plotted.

The threshold length for the initiation of somatic instability at the FRDA locus coincides with the length of a eukaryotic Okazaki fragment

Using SP-PCR, we previously showed that the threshold length for the initiation of somatic instability in vivo is between 26 and 44 uninterrupted GAA triplet repeats (14). Here, using the same technique, we analyzed a total of 5593 individual FRDA molecules with constitutional allele sizes ranging from 8 to 66 uninterrupted GAA triplet repeats to more precisely define the threshold length. SP-PCR of (GAA)8–20 (n = 349 molecules), (GAA)30 (n = 560 molecules) and (GAA)39 (n = 1150 molecules) showed complete stability (Figure 4A and B) (data not shown). In contrast, SP-PCR analysis of (GAA)44 (n = 2304 molecules) and (GAA)66 (n = 1230 molecules) showed somatic instability in vivo, with mutation loads of 6.3 and 30%, respectively (Figure 4C and D). This indicates that the threshold length for the initiation of somatic instability in peripheral leukocytes in vivo at the FRDA locus is between 40 and 44 uninterrupted triplet repeats. The (GAA)39 allele, which showed no somatic instability (Figure 3B), is known to have undergone hyperexpansion in one of three germline transmissions, producing a (GAA)650 allele in the offspring. Therefore, it seems that the minimum length required for the initiation of somatic instability at the FRDA locus may be longer than that required for germline instability.

Figure 4.

The threshold length for the initiation of somatic instability of the GAA triplet repeat at the FRDA locus is between 40 and 44 uninterrupted triplets. SP-PCR was performed on 5593 individual FRDA molecules, with allele sizes ranging from 8 to 66 uninterrupted GAA triplet repeats, i.e. spanning the normal and PM allele range. Representative Southern blots of SP-PCR amplifications are shown. (A and B) SP-PCR analysis of (GAA)30 and (GAA)39 alleles showed complete stability in vivo. (C and D) SP-PCR analysis of (GAA)44 and (GAA)66 showed somatic instability in vivo.

(GAGGAA)n hexanucleotide interruptions stabilize the (GAA•TTC)n triplet repeat at the FRDA locus in somatic cells in vivo

To test whether (GAGGAA)n hexanucleotide interruptions in (GAA•TTC)n repeat tracts cause stabilization of triplet repeats, as has been observed in E.coli (44) and predicted in human germline transmissions (3), SP-PCR analysis was performed using leukocytic DNA from carriers of two similar (GAA•TTC)n alleles. One carrier had a pure (GAA)107 allele, and another a (GAA)114* allele interrupted with a (GAGGAA)5 hexanucleotide sequence close to the 3′ end of the repeat tract [(GAA)76(GAGGGA)(GAA)18(GAGGAA)5(GAA)8]. SP-PCR of 1316 individual FRDA molecules from the carrier of the pure (GAA)107 allele revealed 294 variant bands (mutation load = 22.3%), ranging in size from 63 to 136 triplets (Figure 5A). In contrast, SP-PCR analysis of 1294 individual FRDA molecules from the carrier of the interrupted (GAA)114* allele showed no somatic variability (Figure 5B). This indicates that the (GAGGAA)n hexanucleotide interruption, which is frequently seen in FRDA alleles with >27 triplets (3), stabilizes the repeat tract in somatic cells in vivo. This is an interesting result since the hexanucleotide interruption maps close to the 3′ end of the (GAA)114* allele, leaving a tract of 76 uninterrupted GAA triplets, which is greater than the threshold length for the initiation of somatic instability (Figure 4).

Figure 5.

(GAGGAA)n hexanucleotide interruption stabilizes the GAA triplet repeat at the FRDA locus in somatic cells in vivo. (A) Representative SP-PCR amplifications of a (GAA)107 allele containing uninterrupted GAA triplet repeats at the FRDA locus showed somatic instability. (B) Representative SP-PCR amplifications of a (GAA)114* allele interrupted by a (GAGGAA)5 hexanucleotide sequence showed no appreciable somatic instability.

(GAGGAA)n hexanucleotide interruptions stabilize the (GAA•TTC)n triplet repeat when propagated in plasmids in E.coli

In order to test whether the (GAGGAA)n interruption also stabilizes (GAA•TTC)n repeats in E.coli, we cloned PCR products obtained from leukocytic DNA of the two individuals mentioned above, one having a pure (GAA)107 allele, and another having an interrupted (GAA)114* allele. The resulting PCR products were inserted into pUC19 in both orientations relative to the ColE1 origin of replication, generating the following constructs: (GAA)111, (GAA)101*, (TTC)108 and (TTC)95* (see Materials and Methods for exact sequences). Bacterial growth curves were determined for each of the four constructs as an indication of plasmid replication (as shown in Figure 2). The pure (GAA•TTC)n repeat resulted in slower growth when GAA was the template for lagging strand synthesis [compare (GAA)111 with (TTC)108; Figure 6A], as was previously observed with shorter repeat tracts (Figure 2B). However, comparison of (GAA)111 versus (GAA)101* revealed similar growth patterns, indicating that plasmid replication was similarly impeded by the interrupted GAA tract. Despite the similar patterns of bacterial growth observed for pure and interrupted GAA constructs, the pure repeat sequence was found to be significantly more unstable than the interrupted repeat sequence in both the GAA (92 versus 31.9%, P < 0.001) and TTC (29.6 versus 7.6%; P = 0.004) orientations (Figure 6C), and this instability was composed almost entirely of contractions (data not shown). Interestingly, the stabilizing effect of the hexanucleotide interruption was more pronounced in the GAA orientation (Figure 6C). In the TTC orientation, the (GAGGAA)n interrupted repeat tract was very stable (Figure 6C), similar to the result we observed for this sequence in human somatic cells in vivo (Figure 5B).

Figure 6.

(GAGGAA)n hexanucleotide interruptions stabilize the (GAA•TTC)n triplet repeat, despite slower bacterial growth, when propagated in plasmids in E.coli. All error bars reflect ± SD. (A) Growth curves generated from OD600 measurements at each time point from 9 to 24 h of growth, in triplicate, are shown for E.coli containing pure GAA-111 (filled diamond) and pure TTC-108 (filled square) plasmids. (B) Growth curves generated from OD600 measurements at each time point from 10–24 h of growth, in triplicate, are shown for E.coli transformed with interrupted GAA-101* (filled diamond) and pure GAA-111 (filled square) plasmids. (C) Repeat instability was determined for pure and interrupted (asterisk) (GAA•TTC)n repeat sequences in both orientations after 22 h of culture. The bar graph shows the percentage of colonies containing repeat tracts of altered length (% instability). GAA-111 and TTC-108 were significantly more unstable than GAA-101* and TTC-95*, respectively. The stabilizing effect of the hexanucleotide interruption was more pronounced in the GAA orientation.

DISCUSSION

Friedreich ataxia is a recessive disease and asymptomatic heterozygotes, i.e. individuals who have one FRDA allele containing a fully expanded repeat, represent 0.5–1% of the Indo–European population (2,4). Consequently, most Friedreich ataxia patients inherit fully expanded alleles (typically containing 600–1200 triplets). Hyperexpansion of PM alleles as a means of inheriting the FRDA mutation occurs very rarely (3,4,12,13). This is in contrast to all the other triplet repeat diseases, which are dominantly inherited, wherein de novo expansions are much more frequent. We therefore believe that the reversal of a fully expanded allele to the normal size in somatic cells, rather than prevention of germline hyperexpansion, represents an appropriate strategy for the treatment of Friedreich ataxia. We have previously shown that, unlike the expansion bias of CTG repeat in myotonic dystrophy (45–50), fully expanded GAA triplet repeat alleles at the FRDA locus have a marked tendency to contract in somatic cells in vivo, in some cases even reverting to the normal/premutation size range (14). Our goal is to understand the molecular mechanism(s) underlying the apparently spontaneous reversion of the FRDA mutation, and to devise methods to accelerate this process in somatic cells.

Here, we show that (GAA•TTC)n repeats are more unstable when GAA is the template for lagging strand synthesis during plasmid replication in E.coli, with a marked contraction bias. Ohshima et al. (6) had previously noted increased smearing of their cloned inserts upon multiple rounds of replication, indicative of a contraction bias in plasmids propagated in E.coli when GAA was the template for lagging strand synthesis. During the preparation of this manuscript, a paper was published reporting similar results in a yeast model system (37). The instability of the (GAA•TTC)n sequence was orientation-dependent, with increased instability and a contraction bias when GAA was the template for lagging strand synthesis. These data support our conclusion that (GAA•TTC)n repeat instability is replication-mediated, and also indicate that the mechanism of the strand-specific, differential instability is conserved in prokaryotes and eukaryotes, as is seen for instability of CNG repeats in bacteria and yeast (17–26). Our present analysis of individual replication events using the colony PCR method allowed us to precisely measure the degree and type of instability in the E.coli model system. Furthermore, it allowed us to accurately visualize the spectrum of mutations that occur after individual replication events. Our analysis revealed that it is mainly large contractions that increase in frequency when GAA is the template for lagging strand synthesis. Large contractions frequently resulted in complete reversion to the normal size range. Moreover, in peripheral leukocytes derived from human carriers of similar sized alleles, we identified the same spectrum of mutations. Somatic mutations in vivo consisted mainly of small slippage events and large contractions into the non-disease size range. The similarity of the mutational spectra observed in multiple independent systems, especially the observation of large contractions, suggests that a common mechanism may underlie the instability of (GAA•TTC)n repeats.

The mechanism(s) responsible for GAA triplet repeat instability are poorly understood. We propose that at least two distinct mechanisms underlie the mutations we observed. Small length changes of the repeat tract (<10% variation) may occur as a result of slippage and mispairing during replication of the triplet repeat. The equal frequency and magnitude of small length changes in both ‘GAA’ and ‘TTC’ orientations is consistent with the expectation that slippage is equally likely to occur during leading or lagging strand synthesis, and with the observation that GAA and TTC strands undergo approximately the same degree of slippage during rolling circle replication (51). However, our results implicate erroneous lagging strand synthesis, when GAA is the template strand, as the likely mechanism for the generation of large contractions of the (GAA•TTC)n repeat (Figure 7). The incremental accumulation of large contractions was associated with DNA replication when GAA was the template for lagging strand synthesis. Our in vivo data show that the threshold length for the initiation of somatic instability is between 40 and 44 uninterrupted triplets. Therefore, somatic instability in vivo may initiate when the length of the (GAA•TTC)n repeat exceeds the length of a eukaryotic Okazaki fragment. If the single-stranded lagging strand template were to adopt a stable or metastable secondary structure(s), the replication machinery could bypass a variable number of repeats in the nascent strand, resulting in contraction of the repeat (Figure 7). The 50-fold lower affinity of RPA for polypurine sequences (34) and the selective ability of GAA sequences to adopt intrastrand structures (17,35,36) lend further support to this mechanism. The frequent observation of large contractions and paucity of intermediate-sized contractions could stem from a minimum length requirement for GAA repeats to maintain a stable structure in order to be bypassed in the nascent strand. These data have important implications for understanding the molecular basis of somatic instability at the FRDA locus.

Figure 7.

Model depicting the genesis of large contractions during lagging strand synthesis of GAA triplet repeat sequences (in eukaryotic replication). ‘H’, helicase; ‘PCNA’, proliferating cell nuclear antigen; RPA, replication protein A. Polδ (large rectangle) replicates the leading strand at the advancing fork. Polα/primase (gray filled circle) initiates Okazaki fragment synthesis, and polδ elongates the nascent Okazaki fragment during lagging strand synthesis. The single-stranded lagging strand template is bound by RPA). Non-repeat sequence and the complementary TTC repeat sequence are shown as solid black lines, and the GAA triplet repeat is shown as a dotted line. When the GAA triplet repeat sequence expands beyond the length of an Okazaki fragment (the threshold length of 40–44 triplets at the FRDA locus), individual fragments would have to be initiated, elongated and ligated within the length of the repeat tract. We propose that the 50-fold reduced affinity of RPA for purine-rich sequences [compared with pyrimidine-rich sequences (34)] would allow the single-stranded GAA strand to adopt stable/metastable secondary structures (‘?’), which would result in bypassing of a variable number of GAA repeats in the nascent Okazaki fragment, thus resulting in the experimentally observed contractions. Large contractions (and the under-representation of intermediate sized contractions) in vivo involving alleles with 78–105 repeats could stem from the minimum length required for the secondary structure(s) to be stable.

It is likely that a structural transition may underlie the initiation of somatic instability in vivo. The minimum length for the formation of sticky DNA (59 triplets) (30) is greater than the 40–44 triplet repeat threshold for the initiation of somatic instability. However, Potaman et al. (32) showed that the (GAA•TTC)n repeat undergoes a structural transition from a stable intramolecular triplex at 9–23 triplets to an intramolecular bi-triplex structure at 42 triplets, coinciding in length with the threshold for the initiation of somatic instability in human cells. Interestingly, the (GAA)114* hexanucleotide interrupted allele was stable in somatic cells, despite containing a pure repeat tract of 76 triplets. Pure (GAA)111 and (TTC)108 sequences were also significantly more unstable than interrupted (GAA)101* and (TTC)95* sequences, respectively, when propagated in E.coli. However, the interruption appeared to have no effect on the slowed bacterial growth. The mechanism by which the hexanucleotide interruption confers stability is unknown, but it may be due to the inability of the impure GAA template strand to adopt a stable secondary structure. Previous work has shown that a repeat tract comprised entirely of (GAGGAA)n repetitive sequence (50% non-GAA) does not form sticky DNA, unlike pure (GAA)n repeat sequences (44). Interestingly, in the plasmid replication model the hexanucleotide interruption resulted in significantly enhanced stability of the adjacent repeat when GAA was the lagging strand template, despite interfering with efficient plasmid replication. This dissociation of replication blockade and repeat instability suggests that while bacterial growth and replication may be slowed by the homopurine•homopyrimidine nature of the sequence, purity of the triplet repeat tract is required for GAA instability.

Furthermore, it seems that (GAA•TTC)n repeats are quantitatively more unstable than (CTG•CAG)n and (CGG•CCG)n repeats in somatic cells in vivo. SP-PCR analysis of similar-sized alleles of the other triplet repeats in leukocytes revealed very low mutation frequencies compared with the mutation loads we observed with (GAA•TTC)n alleles (50,52). The reason for these differences is not known, but it is interesting to note that during the evolution of the human genome, (GAA•TTC)n repeats have undergone significant instability as reflected by the development of a wide range of allele lengths in comparison with (CTG•CAG)n and (CGG•CCG)n sequences (53).

In conclusion, we have shown that the (GAA•TTC)n repeat that causes Friedreich ataxia is destabilized during DNA replication, undergoing large contractions when GAA serves as the lagging strand template. In somatic cells in vivo, the (GAA•TTC)n repeat at the FRDA locus initiates instability between 40 and 44 triplets, displays significant mutation load, and the mutational spectrum includes large contractions that result in reversion to non-disease alleles. These data indicate that the FRDA mutation is reversible.

We are grateful to the patients and their families for participating in this study. We would like to thank Dr Gillian Dalgliesh for critically reviewing this manuscript. This research was supported in part by grants from the NIH/NINDS (NS047596), American Diabetes Association and OCAST to S.I.B. S.S. was supported by a fellowship from the Presbyterian Health Foundation.

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Author notes

1Department of Biochemistry and Molecular Biology and 2Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, 3Bruce Leroy Centre for Genetic Health Research, Murdoch Children's Research Institute, 4Department of Pediatrics, University of Melbourne, Australia, 5BioGeM Consortium, 6IEOS-CNR and 7DBPCM, Federico II University, Naples, Italy and 8Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, USA

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