Instability of a Premutation-Sized CGG Repeat in FMR1 YAC Transgenic Mice

doi:10.1006/geno.2002.6849, available online at http://www.idealibrary.com on IDEAL

Article

Instability of a Premutation-Sized CGG Repeat in FMR1 YAC Transgenic Mice Andrea M. Peier and David L. Nelson* Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA *

To whom correspondence and reprint requests should be addressed. Fax: (713) 798-1116. E-mail: [email protected].

Fragile X syndrome results from the massive expansion of a CGG repeat in the 5⬘ untranslated region of the gene FMR1. Data suggest that the hyperexpansion properties of FMR1 CGG repeats may depend on flanking cis-acting elements. We have therefore used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that carried repeats of varying lengths and amounts of flanking sequence. Lengthdependent instability in the form of small expansions and contractions was observed in both male and female transmissions over five generations. No parent-of-origin effect or somatic instability was observed. Alterations in tract length were found to occur exclusively in the 3⬘ uninterrupted CGG tract. Large expansion events indicative of a transition from a premutation to a full mutation were not observed. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and that in human. Key Words: fragile X syndrome, triplet repeat sequence, yeast artificial chromosome, mouse model, dynamic mutation

INTRODUCTION Over the past decade the pathogenesis of many human genetic diseases has been attributed to a group of dynamic mutations—expansions of trinucleotide repeats [reviewed in 1]. Fragile X syndrome is a common form of inherited mental retardation affecting 1 in 3000–4000 individuals [2]. Most mutations causing this disorder result from expansion of a CGG trinucleotide repeat in the 5⬘-untranslated region (UTR) of the gene FMR1 [3]. As a consequence of this expansion, transcription of the FMR1 locus is repressed due to abnormal methylation, leading to loss of protein (FMRP) expression and resulting in the disease phenotype [4,5]. In the human population the repeat is normally polymorphic, ranging from 5 to 50 triplets. Affected individuals carry full mutations, which consist of repeat lengths > 200 and can reach lengths of over 1000 triplets. A third class of alleles is intermediate in size (50–200 repeats). These are benign and have been termed “premutations.” Whereas normal alleles are stably transmitted to offspring, premutations are highly unstable and expand with a high frequency to a full mutation in transmissions from females [6,7]. The molecular mechanisms mediating the instability of the CGG repeat remain elusive. However, certain factors are believed to influence the behavior of these sequences. Despite

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the high mutation rate in this disease (estimated to be 0.8 ⫻ 10–4), maintenance of linkage disequilibrium with dinucleotide repeat markers within 150 kb of the repeat is observed [8,9]. As some haplotypes show an increased association with the fragile X mutation, investigators have hypothesized that cisacting elements may increase the likelihood of instability of these sequences. Several studies have shown that the CGG tract contains one or more AGG interruptions at an interval of every 9 or 10 CGGs [10–12]. One element in cis is the length of the uninterrupted repeat, which has been demonstrated to influence stability. Transmission studies of premutation and unstable alleles in human pedigrees reveal a correlation between the length of pure repeat tracts and the propensity for instability, with unstable transmissions beginning with allele lengths of 35 repeats [10]. Interestingly, changes in repeat length have been shown to exhibit a polarity that occurs exclusively at the 3⬘ end of the uninterrupted repeat [10,13]. Most attempts to mimic trinucleotide repeat instability in mice have focused on the CAG triplet repeat disorders. Initial studies with transgenic mice carrying disease-associated alleles found the CAG repeat tracts to be stable within the context of cDNA-based constructs [14–16]. More recently, by using longer repeats and/or including the repeats within their native DNA context, instability has been observed in the mouse [17–20]. Fewer studies of CGG repeats have been done

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FIG. 1. Modification of YAC209g4c. A premutation-sized CGG repeat was subcloned into a yeast integration plasmid (pCGG-EX), linearized, and transformed into yeast containing the YAC 209g4c with 20 CGGs. Integration of the plasmid into the FMR1 locus results in a duplication of the repeat region and also allows growth in medium lacking uracil. PCR and Southern blot analysis were used to evaluate the correct pop-in event. Subsequent plating of transformants on non-selective medium (Ura+) results in a spontaneous excision event (pop-out) of the duplicated region which results in retention of the parental sized repeat or replacement with the premutation sized allele.

RESULTS

in mice. These groups generated transgenic mice carrying premutation-sized CGG tracts inserted within the mouse genome [21], within the context of the cDNA [22], or within the 5⬘ part of FMR1 [23]. Results revealed that repeat tracts were stable in mice at lengths that show significant levels of instability in humans. Recent advances have allowed the generation of transgenic mice by inserting yeast artificial chromosomes (YACs) carrying large fragments of exogenous DNA [reviewed in 24]. The large size capacity of YACs, up to 1 Mb, allows the inclusion of distant regulatory and intragenic sequences that may be critical for proper expression. Additionally, YACs are extremely amenable to genetic manipulation by homologous recombination in yeast, permitting the efficient introduction of mutations, including insertions, deletions, and nucleotide substitutions, into precise locations within the YAC insert. Several studies investigating globin gene regulation have successfully used “wild-type” and mutant YACs carrying the globin gene locus to recreate human globin developmental mutants [25]. La Spada et al. introduced a large CAG repeat into the androgen receptor locus on a YAC in order to model spinobulbar muscular atrophy (SBMA) [26]. We have used a YAC-based strategy to develop a mouse model to study instability of the fragile X repeat. Given the murine instability data for the CAG repeats and the hypothesis that cis-acting elements are important for stability of the FMR1 locus, we used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that varied in both repeat length and flanking sequence. We observed CGG length-dependent instability beyond that seen in other mouse models containing CGG repeats with cDNA-based constructs. These mice exhibited intergenerational instability represented by expansions and contractions that reproduce some of the features observed in human transmissions.

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Modification of YAC 209g4c An in situ expansion corresponding to a premutation-sized allele was introduced into a previously characterized 450-kb YAC that spanned the human Xq27.3 region [3,27]. YAC 209g4c contains the entire human FMR1 gene with 20 CGG repeats and approximately 300 kb upstream of the FMR1 start site and 100 kb downstream. We used a two-step gene replacement method, termed the pop-in/pop-out procedure, that uses the gene URA3 for both positive and negative selection [28] (Fig. 1). Because the YAC vector carries URA3 on one of its vector arms, the YAC was first retrofitted with pRV1 [29], replacing the URA3 locus with the auxotrophic marker LYS2 and a neomycin resistance gene (neo) for cell culture studies (data not shown). This modified YAC was designated YapRV.2. A (CGG)92 allele was isolated from an adult male carrying a premutation inherited from a premutation male with the configuration (CGG)9AGG(CGG)9AGG(CGG)72. This allele, when transmitted through the female germ line in humans, would be expected to expand to a full mutation at a high frequency [6,7]. Indeed, this allele did show expansion to the

FIG. 2. PCR analysis of potential founders for the expanded CGG repeat at the YAC FMR1 locus. PCR was performed with primers Fu-c and Fu-f, which also amplify the endogenous mouse locus (corresponding to a 245-bp PCR product). The presence of the (CGG)90 repeat is indicated as a 490-bp PCR product. Representative results are presented for five of the nine founders (lanes 1–5), wild-type non-transgenic DNA (lane 5), and the modified YAC (lane 6) (YapRV-EX). Products were resolved on an ethidium-stained agarose gel.

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FIG. 3. Extent of flanking sequence present in YAC transgenic animals. (A) Diagram of YAC209g4c containing the entire FMR1 gene. The order and relative location of sequence tagged sites (STSs) spanning the length of the YAC used for screening potential founders are included and designated by a filled circle. (B) STS content analysis of the FMR1 YAC transgenic mice. For each of the transgenic lines results of the PCR screening are given as follows: +, present; -, absent.

Article

A

B

full mutation upon transmission from the individual’s daughter. The CGG repeat region, in addition to several hundred base pairs of flanking sequence, was cloned into a yeast integration plasmid (YipCGG-EX). These vectors lack an autosomal replication sequence (ARS) and therefore cannot be maintained autonomously in the cell without integration into the yeast genome. The construct was linearized and transformed into yeast carrying the retrofitted YAC, resulting in the integration of the plasmid sequence flanked on each side by the repeat. Amplifications by PCR, using primers that flank the CGG repeat, and Southern blot, using probes within the duplicated region, were used to screen the resulting URA+ colonies for correct pop-in events (data not shown). Positive transformants were then plated onto non-selective medium (URA+), which allows the spontaneous excision of the plasmid sequence due to an intrachromosomal recombination event between the repeat regions. This pop-out event results in either retention of the parental-sized repeat or replacement with the expanded allele. Colonies were screened by PCR for the expanded allele and further characterized by Southern blot using an exon 1 probe as well as a vector probe to ensure that no rearrangements had occurred. Additionally, agarose plugs were prepared from yeast cultures carrying the modified YACs and subjected to pulse-field gel electrophoresis. Gels were blotted and Southern analysis further demonstrated that no gross rearrangements, deletions, or insertions had occurred (data not shown). YACs carrying various repeat sizes were identified, including one with 90 CGGs (YapRV-EX). Sequencing of the repeat confirmed the presence of the two AGG interruptions and that no additional alterations had been introduced (data not shown). Generation of YAC Transgenic Mice Purified YapRV-EX YAC DNA was microinjected into fertilized FVB/N (FVB) mouse oocytes and transplanted into foster mothers. Potential founders were initially analyzed by

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PCR using primers specific for the CGG repeat locus. Nine founders carried the human FMR1 CGG repeat (Fig. 2). Extensive attempts at breeding showed that four founders did not transmit the YAC sequence, most likely due to mosaicism in the germ line of these mice. Four animals were found to be positive for the entire FMR1 gene (TG7, TG10, TG481, and TG484) and significant flanking sequences by PCR analysis using sequence tagged sites (STSs) that spanned the length of the YAC (Fig. 3). One founder (TG10) was sterile and did not breed. TG296 carried the CGG90 allele, but only ~ 5 kb of flanking sequence. Characterization of (CGG)90 Transgenic Mice The CGG repeat length was determined by PCR using primers (Fu-c and Fu-f) [7] that amplify both the endogenous mouse Fmr1 locus and the human repeat. Radiolabeled PCR products were separated on 5% polyacrylamide gels. The repeat varied from 14 to 110 CGGs in the transgenic mice, which was presumably due to instability of the repeats in the yeast host (A.M.P. and D.L.N., manuscript in preparation). Initial analysis of mice carrying a small size CGG repeat (TG7 and TG481) showed no instability and therefore subsequent focus was placed on analysis of the two transgenic lines harboring the premutation-sized repeat (TG296 and TG484). Table 1 summarizes the CGG repeat lengths and instability frequency for each of the transgenic lines analyzed. The TG484 line carried two repeat sizes (CGG90 and CGG33) that co-segregated in subsequent generations. Southern blot analysis and fluorescence in situ hybridization (FISH) of this line showed that the YAC DNA integrated at a single locus and contained either three or four copies of the repeat sequence (data not shown). Additionally, the TG484 transgenic line carried at least two copies of the (CGG)90 array because alterations in tract length detected by PCR always exhibited a (CGG)90 repeat in addition to the array that had undergone a change. We also investigated whether both alleles were transcribed in the TG484 line using

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TABLE 1: FMR1 YAC transgenic lines TG line

Copy number

Total progeny

TG positive progeny

CGG repeat length

TG10*

ND

–

–

110, 90

Types of changes

Mutation frequency –

TG7

ND

154

77

38

0

TG481

2–3

68

35

20, 14

0

TG296

1

471

223

90

+1, +2, –1, –2

16.1%

TG484

3–4

754

381

90, 33

+1, +2, +3, +4

15.5% (for CGG90)

–1, –2, –3, –15

0% (for CGG33)

*TG10 did not breed.

RT-PCR. RNA was extracted from mouse brain, and following the initial first-strand cDNA synthesis, primers specific for the CGG repeat were used to amplify FMR1 message (Fig. 4). These results demonstrated that both the (CGG)90 and (CGG)33 repeats were transcribed for the TG484 line. Western blot analysis also confirmed that transgenic human protein was produced [30]. The TG296 line contained a single copy of the (CGG)90 repeat. PCR with nearby STSs demonstrated the presence of YAC sequences ~ 4 kb upstream and ~ 1 kb downstream of the CGG tract. Southern blot analysis using methylation-sensitive restriction enzymes demonstrated that the CpG island 5⬘ to the repeat was unmethylated in both transgenic lines (data not shown). Intergenerational Instability of the CGG Repeat in FMR1 Transgenic Mice The TG484 and TG296 lines were bred to wild-type FVB mice for five generations. We observed the first tract length change in the second-generation (F2) for the (CGG)90 repeat in both transgenic lines. Although the (CGG)33 allele present in line TG484 did not exhibit instability, we did observe that the (CGG)90 repeat was altered in length in 64 of 381 transmissions (16.8%; Table 1). During the F2 generation, an increase of one triplet (CGG91) occurred in animal 828 in the TG484 line (Fig. 5). This allele was stably transmitted to the next generation (F3). An F3 male (1284) was then crossed to a wild-type female and the progeny from this litter revealed a female (1960) harboring a contraction of three repeat units (CGG88). Subsequent breeding of 1960 yielded three transgenic offspring, of which two pups contained the parental size repeat (CGG88) and the third pup (2354) showed an expansion of one repeat giving rise to a repeat length of (CGG)89. These data indicate that instability not only occurred but also continued through successive generations. Figure 5 also presents instability in a pedigree from the TG296 line. Alterations in CGG repeat length were observed in 38 of 223 transmissions (or 17%) in the TG296 transgene positive progeny. These frequencies of intergenerational instability are significant in comparison with previous murine studies using similar-sized CGG repeat lengths whereby transgenic mice carrying a (CGG)120 tract did not show a change in repeat length over four generations in a total of 62 transmissions [23].

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Magnitude and Types of Changes Most alterations involved subtle changes of one to a few CGGs. In no case did we observe a large expansion akin to the transitions to full mutations that occur in humans when an allele of this size is inherited from premutation females. We found expansions were significantly more prevalent than contractions in both TG484 (P < 0.0001) and TG296 (P < 0.002) transgenic lines. The size of the expansions ranged from +1 triplet to +4 triplets, whereas contractions proved to encompass a wider size range (–1 CGG to –15 repeats). The distribution of the size changes in the TG484 line showed greater variation compared with that of the TG296 line. No Effect of Parental Sex on Repeat Instability We also calculated the mutation frequency in each line from crosses with only one transgenic parent in order to identify the parental origin of the repeat change. For line TG296, we observed 32 repeat length changes in 193 transmissions and for line TG484, we detected 55 alterations in tract length in 323 transmissions (Table 2). The frequencies of instability for TG296 and TG484 were similar (16.6% versus 17%). No sig-

FIG. 4. RT-PCR analysis of the TG484 line. RNA was extracted from total brain tissue from two different TG484 transgenic animals. Before the first-strand synthesis reaction, RNA was treated with DNase. PCR was performed using primers specific for the CGG repeat. Lanes 1 and 3 show the PCR products amplified from the (CGG)33 and (CGG)90 allele in the RT+ reaction. Lanes 2 and 4 are the controls carried out without reverse transcriptase.

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nificant difference in the frequency of instability was observed between male and female transmissions. A maternally inherited premutation-sized repeat was unstable in 15.2% of the female transmissions, whereas 17.4% of the progeny inherited an unstable allele when the transgenic parent was a male. Both male and female progeny inherited mutations from the transgenic parent with an equal frequency. The (CGG)90 Repeat Does Not Exhibit Somatic Mosaicism Somatic instability of the premutation-sized repeat was assayed by performing PCR on the DNA derived from brain, testis, heart, spleen, kidney, and liver tissue in at least one mouse from each line. No evidence of mosaicism was detected in the transgenic tissues analyzed (data not shown). No Effect of Parental Age on Instability We also investigated the effect of parental age on instability by comparing mutation frequencies between parents greater than and less than 6 months old. No significant effect of age was observed for either parent or transgenic line. Line TG484 for example, showed 16% instability in the transmissions from a parent > 6 months old versus 16.4% of the transmissions from a parent < 6 months in age. Analysis of transgenic progeny from a parent as a function of age also showed no differences in the frequency of instability as the parent aged. Similar findings were observed for the TG296 line. Thus, in these experiments, CGG repeat instability does not seem to be influenced by the age of the parent. Both the somatic data and the parental age data presented here differ from previously reported mouse models carrying large CAG/CTG repeat tracts [19,31,32]. Polarity of Changes In humans, alterations in the repeat tract at the FMR1 locus have shown a polar bias whereby changes in repeat length occur at the 3⬘ end in the uninterrupted tract in most cases analyzed. We investigated whether changes in the premutation-sized allele in the transgenic mice were likewise occurring at the 3⬘ end by determining the AGG interspersion pattern of the repeat tracts in mice harboring a change. Our group and others [10,33] have shown that the AGG interspersion pattern of the FMR1 repeat allele can be determined by MnlI restriction enzyme analysis. Briefly, PCR products containing the CGG repeat are digested with MnlI, whose restriction recognition site contains an AGG and cuts several nucleotides near to the interruption. Digested PCR products are run on high-percentage agarose gels, blotted, and hybridized with a CGG oligonucleotide probe. A specific pattern of bands is generated depending on the size of the repeat and the pattern of AGG interruptions. This analysis was performed on genomic DNA from both the TG484 and TG296 lines with animals harboring tract length alterations. Figure 6 shows representative data. Results revealed that the (CGG)33 repeat in the TG484 line was a pure CGG tract lacking AGG interruptions. Both “parental” AGGs were present in the (CGG)90 alleles.

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TABLE 2: Trinucleotide repeat instability in (CGG)90 transgenic mice Sex of parent

Alterations/transmissions* (rate of instability)

Combined instability

TG296

TG484

10/73

17/104

27/177

(13.7%)

(16.3%)

(15.2%)

8/73

15/104

23/177

(11%)

(14.4%)

(13%)

2/73

2/104

4/177

(2.7%)

(1.9%)

(2.2%)

Male total

22/120

38/219

60/339

(18.3%)

(17.4%)

(17.7%)

Expansions

17/120

28/219

45/339

(14%)

(12.8%)

(13.3%)

Contractions

5/105

10/219

15/339

(4.3%)

(4.6%)

(4.4%)

32/193

55/323

87/516

(16.6%)

(17%)

(16.9%)

25/193

43/323

68/516

(13%)

(13.3%)

(13.2%)

7/193

12/323

19/516

(3.6%)

(3.7%)

(3.7%)

Female total Expansions Contractions

Both total Expansions Contractions

Similar rates of instability were observed for both transgenic lines and for both female and paternal transmissions. *Transmissions are calculated from crosses with only one transgenic parent.

Changes in repeat length occurred at the 3⬘ end as no alterations in the fragments for the two 5⬘ MnlI fragments were observed. Hybridization with flanking oligonucleotides detected fragments of the expected size, which further confirmed that variation in allele length was due to changes in the 3⬘ most CGG repeat containing the MnlI fragment (data not shown).

DISCUSSION We have developed a mouse model to study instability of the fragile X repeat. As transgenic mice with premutation-sized CGG repeats introduced on constructs carrying little genomic sequence did not show instability, our strategy was to generate transgenic mice carrying a premutation-sized repeat on a YAC in order to study the behavior of these sequences within their native chromosomal context. An in situ CGG expansion corresponding to a premutation-sized allele was introduced into a human YAC carrying the entire FMR1 gene.

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Article A

doi:10.1006/geno.2002.6849, available online at http://www.idealibrary.com on IDEAL

B

FIG. 5. PCR analysis of the CGG repeat in FMR1 YAC transgenic mice. (A) Pedigree results for the TG484 line. Mouse 1284 exhibits an increase of +1 triplet (CGG)91 in addition to the (CGG)90 allele. This allele exhibited instability in the next generation with mouse 1960 carrying a contraction of four CGGs (CGG)87. A litter from this transgenic female resulted in two animals retaining the parental sized repeat (2355 and 2359) and a third pup harboring a +1 expansion (2354) with a new repeat length of (CGG)89. (B) Pedigree results for the TG484 and TG296 line. Animal 1278 carries an expanded allele of +2 CGG triplets. This allele is stably passed on to the female progeny (2476). Mouse 694 is a transgenic from the TG296 line carrying the (CGG)90 repeat. This allele undergoes instability in the next generation as a contraction (1831) and also as expansions (1833, 1834). AS is a premutation male carrying a 92 CGG repeat.

Several lines of transgenic mice were generated carrying repeat sizes ranging from 14 to 110 triplets and also carrying varying amounts of flanking sequence. We observed lengthdependent instability whereby mice harboring small repeat tracts (≤ 33 CGGs) did not show changes in repeat length. However, transgenic mice carrying a (CGG)90 repeat showed moderate intergenerational instability in both male and female transmissions over five generations. These alterations occurred exclusively in the 3⬘ uninterrupted CGG tract. Large

expansion events indicative of a transition from a premutation to full mutation were not observed. The transgenic lines did not show somatic instability or a parental age effect. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and humans and the behavior of the CGG repeat sequence versus CAG repeat tracts in mice. Although somatic instability has been reported for mouse models of myotonic dystrophy (DM) [17,18] and Huntington’s disease (HD) [34,35], we did not find somatic variation of the CGG repeat in tissues isolated from both the TG484 and TG296 transgenic lines. This finding is indeed consistent with the limited somatic instability that has been observed in tissues from humans carrying large repeat tracts. A previous study [36] showed somatic instability in single sperm from premutation males, but the sensitivity of our analysis would not reflect such changes.

A

B

FIG. 6. AGG interspersion analysis of the human CGG repeats in TG296 and TG484 transgenic mice. Lanes 1 and 6–10 represent progeny from the TG484 line. Lanes 2–5 and lane 11 are samples from the TG296 line. AS is a male that carries a premutation with the configuration (CGG)9AGG(CGG)9AGG(CGG)72. (A) MnlI digest of FMR1 CGG repeat products. PCR products were amplified with Fu-c and Fu-f, purified, digested overnight, and run on high percentage agarose gels. (B) Hybridization of MnlI digests. MnlI digests were transferred and probed with a (CGG)10 oligonucleotide. The CGG repeat containing fragments will hybridize with differential intensity depending on CGG repeat content. Three MnlI fragments are expected for the (CGG)90 repeat with the configuration (CGG)9AGG(CGG)9AGG(CGG)70: a 58-bp fragment corresponding to the 5⬘ MnlI fragment; a 30-bp band representing the middle MnlI fragment; and a 234-bp band representing the 3⬘ MnlI fragment. Correctly sized bands are observed in all lanes indicating the retention of both AGGs. In the TG484 samples, the (CGG)33 band is represented by a 154-bp band. This size is expected if no AGGs are present within the CGG tract. Lane 5 is a TG296 animal that was sized to carry a deletion of three CGG triplets. Additionally, the sample in lane 11 was sized to have an expansion of four triplets on denaturing polyacrylamide gels. The 234bp band in (A) seems to be larger than the other samples, corresponding to the addition of triplets occurring at the 3⬘ end of the repeat tract.

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Transgenic mice did not show a parental age effect in either male or female transmissions. There is some evidence in families with fragile X syndrome that suggests an effect of maternal age on repeat length. Mornet et al. noted that among siblings with a full mutation, the younger sibling more often inherited a larger expansion [37]. Another study compared fragile X premutation-female transmissions with risk for expansion as a function of maternal age [38]. The authors attributed the putative maternal age effect to selection of smaller alleles within the offspring’s soma over time rather than an event occurring within the mother’s germ line. In contrast to other murine models of CAG trinucleotide repeats for spinocerebellar ataxia type 1 (SCA1) [19,31], HD [34], or spino-bulbar muscular atrophy (SBMA) [26], we did not observe genetic instability to be correlated with parental age. It remains undetermined whether this pronounced age effect is a feature of the respective human disorders or is an intrinsic property unique to the CAG triplet repeat sequence. Parent-of-origin effects were notably absent from our transgenic mice. We observed a similar frequency of instability when the repeat was transmitted through the female or the male germ line. This is in contrast to the behavior of a similar sized repeat in humans, where premutations have shown a much more limited instability upon male transmissions [39]. Additionally, the direction and magnitude of changes were also equally represented in both sexes. These findings differ with results from other trinucleotide repeat mouse models of the human genetic diseases SCA1 [19,31], HD [35], DM [18], and SBMA [26]. Those studies reported differences in the frequency and direction of changes in repeat length with respect to the sex of the transmitting parent. Why was the hyperexpansion phenomenon not observed in our mouse model? There is some evidence suggesting that the repeat copy number threshold for instability may be higher in mice. Disease-associated alleles in humans for many of the CAG/CTG triplet repeat disorders have been reported as either relatively stable or showing moderate levels of instability in transgenic mice. For example, a 45-CAG repeat and a 55-CTG repeat in the context of a YAC carrying the entire androgen receptor [26] and within a 45-kb genomic fragment carrying the myotonic dystrophy protein kinase (DMPK) [17], respectively, showed alterations in ~ 10% of the transmissions. Larger repeat tracts of 115–162 triplets in the DMPK 39-UTR and in HD exon 1 transgenic mice [34] showed changes in repeat length in ~ 60–70% of meioses. These higher instability rates in the germ line are more similar to their respective human diseases but require longer repeat tracts. More recently, Wheeler et al. have shown that the degree of instability correlated with repeat length by comparing the transmissions of different size repeats inserted within the murine HD locus [35]. Their data revealed length-dependent instability similar to that in human but requiring longer CAG tracts. The CGG repeats in our transgenic mice yield levels of instability intermediate to those in the aforementioned studies. Creating

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Article

transgenic mice harboring a full mutation or larger premutation sized repeats, although technically challenging, may prove to better mimic the human locus. Using the YAC transgenic approach, we sought to delineate potential cis-acting regions required for mediating instability. Data from other mouse models of trinucleotide repeat instability demonstrated that instability could be enhanced by the inclusion of additional genomic flanking sequences. Both transgenic lines carrying the (CGG)90 repeat exhibited similar rates of instability despite differing amounts of flanking sequence. The TG296 line harbored one copy of the (CGG)90 repeat flanked with only ~ 5 kb of YAC sequence. The TG484 line, however, carried three or four copies of the FMR1 locus with one copy containing the pure (CGG)33 repeat allele. A minimum of two copies carry the (CGG)90 repeat, as we consistently observed the parental (CGG)90 allele in addition to a second allele exhibiting a change in repeat size. Given the presence of more than one repeat length and multiple copy number in the TG484 line, it is difficult to ascertain the extent of the flanking sequence surrounding each of the (CGG)90 repeats. Additionally, as fragmentation of the YACs can occur during the microinjection process, it is difficult to ascertain the flanking sequence surrounding each of the CGG arrays. However, RT-PCR analysis demonstrated that at least one of the (CGG)90 alleles was transcribed into RNA, indicating that at least one of the premutation-sized repeats is within the context of the FMR1 gene. It is unclear at the present time whether this is the allele exhibiting instability during transmissions. Nevertheless, our data indicate that a premutationsized allele does not exhibit the hyperexpansion properties that would be observed in humans, even within the genomic context of human FMR1. Additionally, hyperexpansion may not have been observed in our transgenic mice, because the genomic sequence flanking the repeats may not be predisposed to instability. Marked linkage disequilibrium has been shown to exist between certain flanking markers and the fragile X mutation. This suggests the presence of predisposed chromosomes marked by the associated haplotypes. The origin of the YAC used in this study is from a chromosome that is not frequently found in fragile X patients (data not shown). As extensive haplotype analysis has demonstrated marked linkage disequilibrium at the FMR1 locus, generating transgenic mice that carry a cloned repeat from a patient or at-risk haplotype may prove to provide enhanced instability. Indeed, CTG repeats exhibited greater levels of instability in mouse models using transgenes derived from affected individuals [17]. Moreover, the phenomenon of hyperexpansion at the FMR1 locus may be dependent on the chromatin and/or nucleosome state that is unique to the X chromosome, especially because long CGG repeats have been shown to assemble poorly into nucleosomes [40]. Additionally, in the present study, we examined instability in hemizygous mice for which the homologous chromosomes cannot pair in the region of the transgene. Further analysis using mice homozygous for the transgene may address this issue.

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Article

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Although numerous studies involving transgenic and knock-in models for the CAG repeats have reported varying degrees of instability, only a handful of studies have examined the behavior of CGG repeats in the mouse [21–23]. Lavedan et al. [23] reported no instability in transgenic mice carrying similar sized CGG repeats using a transgene that harbored a limited amount of upstream flanking sequence. Small-scale changes were also not observed as measured by Southern blot analysis. These differences may very well be attributed to the method of analysis, as PCR was used almost exclusively in this study and such small changes may have been undetectable by Southern blot analysis. While this paper was under review, a report was published by Bontekoe et al. [41] describing instability of a (CGG)98 repeat introduced at the endogenous mouse locus. The authors observed a rate of instability of at least 10% with most changes occurring through male transmissions. However, as found in the present study, no large-scale changes in tract length were reported. The small changes observed in this study are similar to the types of changes reported for CAG instability in mice. This small-scale variation most likely arises by a mechanism different and distinct from the transition of the premutation to a full mutation in humans. Replication slippage, implicated in microsatellite instability, has been proposed to account for these small changes observed for the CAG/CTG repeats in mice and in other model organisms [42]. The massive expansions observed at the FMR1 locus have been proposed to result from the slippage of Okazaki fragments during replication when repeat tracts reach lengths of ~ 70 pure repeats [10]. Why the large changes are not observed in mice is unclear, as the replication/repair machinery seems to be highly conserved between humans and mice. The observed polar variation among the premutationsized repeat in our transgenic lines in the 3⬘ portion of the CGG tract is similar to the behavior of the repeat in humans [10,13]. Polar variation implicates specific mechanisms of mutation and suggests that in the case of FMR1, the AGG triplets that interrupt the repeat influence stability [13]. The fact that replication slippage is not a frequent event in the 5⬘ portion of the CGG tract suggests that the AGG interruptions confer stability to the repeat. These interruptions are thought to function by anchoring the sequence and preventing slippage during replication or disrupting the formation of secondary structures [43]. Gene conversion, meiotic recombination, sister chromatid exchange, and differential fidelity of the template during replication may all lead to polar variation [13]. Because similar rates of instability were observed in two different transgenic lines, our data indicate that the mechanism is due in part to the intrinsic property of the repeat itself. Although the mechanism is not known, the data suggest that this mechanism of mutation is similar (conserved) between species. Our findings indicate that some of the behaviors of the FMR1 CGG repeat are reproduced in the mouse. However, several differences exist. Our data suggest that, as in humans,

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instability is predominantly a result of gametogenesis. Whether these interspecies differences (that is, trans-acting factors) are contributing to the behavior of these repeats allowing mice to be better adept at faithfully replicating these sequences remains to be determined. Perhaps the pattern of expression of the genes involved in these processes is not conserved between species resulting in a selective constraint on repeat size in the mouse. Efforts are currently underway to cross these transgenic mice to other mouse strains mutant in DNA replication or repair, as these genes may influence the behavior of trinucleotide repeat sequences.

METHODS Modification of YAC 209g4c. We targeted CGG repeat expansions into YAC 209G4 by a two-step gene replacement method, using URA3 in a positive selection step followed by a negative selection step. The YAC was first retrofitted to knock out the URA3 locus on the YAC vector arm. A 9.5-kb fragment containing the neo and LYS2 genes from pRV1 was used to transform the host yeast strain (AB1380) carrying YAC 209g4. Transformants were plated on medium lacking lysine and subsequently replica plated on URA– medium. Growth on lysine but not on uracil indicates a recombination event between the pRV1 fragment and the URA3 locus on the YAC. Two clones that were lys+ and ura– were subjected to PFGE, blotted, and hybridized with a neo probe to establish that the YAC had not been grossly rearranged. pCGG-EX was constructed as follows. A 610-bp PCR product containing 92 repeats from a premutation male was subcloned into pCRII (p88.30). A second PCR product partially overlapping the 3⬘ end of the repeat was also subcloned in a pCRII vector (pAPXB). p88.30 was digested with EcoRI and XhoI and pAPXB was digested with XhoI and BamHI. These two digested fragments were ligated to EcoRI/BamHI digested Yiplac211. Restriction mapping confirmed the integrity of the sequence. The construct was linearized with SmaI and transformed into yeast carrying the retrofitted YAC. The pop-in recombination event leads to a duplication of the CGG repeat containing region. Correct integration of the plasmid sequence was distinguished by digesting yeast genomic DNA with PstI (which cuts outside of the duplicated region), Southern blot and hybridizing with a probe containing the FMR1 CGG repeat region. Of 34 clones analyzed, 7 were integrated correctly. Three clones revealed an integration of a large size repeat allele. These clones were streaked on non-selective medium (Ura+) to allow the spontaneous excision of the URA3 containing plasmid. Individual colonies were picked and subsequently streaked on 5-fluoroorotic acid (FOA) containing plates (lethal to cells retaining the URA3 marker), to select for clones that had excised the plasmid sequence. Excision of pYIPlac211 will result in retention of either the parental YAC (CGG20) or replacement with the permutation-sized allele. Three modified YACs were analyzed by pulse-field gel electrophoresis, Southern blot, and restriction digest mapping to confirm that no rearrangements had occurred during this process. One clone, YapRv-EX, was further characterized and used to generate transgenic mice. Sequence of the premutation-sized repeat. The CGG repeat was sequenced using a modified protocol of the thermo-sequenase radiolabeled terminator cycle sequencing kit (Amersham). Both DMSO (5%) and Betaine (2 M) were added to the reaction mixture. Primers 170 and 172 [11] (2 ␮M) were used to sequence the G-rich and C-rich strand, respectively. Generation of YAC transgenic mice. YAC DNA was prepared for microinjection essentially as described [24] with the following modifications. Concentrated YAC DNA was dialyzed in microinjection buffer (10 mM Tris, pH 7.4, 0.1 mM EDTA, 100 mM KCL) for several hours before microinjection. Initial injections using concentrations at 0.5–1 ng/␮l yielded a low number of progeny. We found that a lower concentration of 0.2–0.5 ng/␮l was optimal. Analysis of transgenic mice. Founders were initially screened for the human CGG repeat locus using primer 571 and primer a [44]. Primers designed to detect STSs that span the length of the YAC were also used to determine the

GENOMICS Vol. 80, Number 4, October 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.

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amount of YAC DNA present in the transgenic lines [30]. Transgene copy number was determined by Southern blot hybridization of human, wild-type, and transgenic mouse DNA using a human-specific FMR1 probe. This probe is a 540-bp PCR product encompassing intron 1 from human FMR1, which yields a 5.2-kb band from an EcoR1 digest. Additionally, a 600-bp PCR product using primers LLK96 [45] and LLK192 (5⬘-CTCAAGGCACATCTGATG-3⬘) that span the 3⬘-UTR of mouse Fxr1 was used as an internal control. Southern blots were scanned by densitometry (Molecular Dynamics). The methylation status of the human FMR1 locus was determined as described [7,27] by Southern blot of EcoRI and BssHII mouse genomic digests. Fluorescence in situ hybridization was performed on mouse fibroblasts obtained from tail tissue grown in culture. Cells were harvested according to standard cytogenetic procedures. YAC 209g4c (500 ng) was labeled with digoxigenin-dUTP (Boehringer Mannheim) by nick translation. Hybridization and detection were performed essentially as described [46]. CGG repeat analysis. To robustly amplify distinct permutation-sized alleles by PCR, the following protocol was used. PCR analysis was performed in a 25 ␮l reaction containing 2 M betaine, 0.2 mM dNTPs, 0.4 ␮M of primers Fuc and Fu-f, 5% DMSO, 1⫻ cloned Pfu buffer, and 0.4 ␮l of an enzyme mixture containing 14 ␮l of Klentaq (AB Peptides) and 1 ␮l of native Pfu (Stratagene) polymerase. Cycling parameters were 98⬚C for 5 minutes, followed by 34 cycles at 98⬚C for 1 minute, 65⬚C for 1 minute, 72⬚C for 2 minutes, and a final extension at 72⬚C for 7 minutes. For sizing, the Fu-c primer was end-labeled with [␥-32P]ATP and polynucleotide kinase (NEB) according to manufacturer’s directions and added to the above reaction. PCR cycles were decreased to 28 and products sized on 5% denaturing polyacrylamide gels run at 90 watts for 7–10 hours. To eliminate PCR artifact, analysis was performed at least in duplicate. Repeats were sized to M13 sequence and human control samples with known repeat lengths. Sequence of the permutation-sized repeat. The CGG repeat was sequenced using a modified protocol of the thermo-sequenase radiolabeled terminator cycle sequencing kit (Amersham). Both DMSO (5%) and Betaine (2 M) were added to the reaction mixture. Primers 170 and 172 [11] were used to sequence the G-rich and C-rich strands, respectively. Expression analysis. RNA was extracted from mouse brain tissue using Trizol (Gibco) and treated with DNase following the manufacturer’s instructions. Oligo-dt primed RNA (5 ␮g) was reverse transcribed using Superscript (Gibco). Prepared cDNA (1 ␮l aliquot) was then amplified by PCR using primers Fu-c and Fu-f in a 25 ␮l reaction volume as described above. Western blot analysis was performed essentially as described [30] using total brain protein lysates prepared from TG484 animals. AGG interspersion analysis. Analysis was performed essentially as described [10] with the following modifications. A 50 ␮l PCR reaction using primers 212 and 571 (as described above) was Quiaquick (Qiagen) purified and resuspended in 30 ␮l TE. We subsequently used 20 ␮g PCR product for the MnlI digest, ran it on 3% Metaphor agarose gels, blotted it bidirectionally, and hybridized it with one of four different oligonucleotide probes. A (CGG)10 probe was used and hybridized at 65⬚C; 27711 (5⬘-CACGGCGCCGCTGCCAGG-3⬘) lies upstream of the CGG repeat, is contained within the 5⬘ MnlI fragment, and was hybridized at 45⬚C. The two oligonucleotides downstream of the repeat, Mnl3a (5⬘-CTGGGCCTCGAGCGCC-3⬘) and Mnl3b (5⬘-CGCAGCCCACCTCCTCCGGGGG-3⬘), were hybridized at 56⬚C. Hybridization was carried out overnight in 0.25 M NaPO4, 0.25 M NaCl, 5% SDS, 10% PEG, 1 mM EDTA, and 100 ␮g/ml herring sperm DNA. Blots were washed the following day three times for 30 minutes at the same temperature used for hybridization with 0.05 M NaPO4, 0.5% SDS, and 1 mM EDTA solution and exposed to autoradiographic film for 3 hours to 2 days to allow visualization of the bands.

ACKNOWLEDGMENTS We thank Paul Overbeek and Gabby Schuster of the Baylor College of Medicine Mental Retardation Research Center (MRRC) transgenic mouse core lab for assistance in generating YAC transgenic mice; Jennifer Seifert for animal and technical assistance; and Danna Morris for helpful suggestions for yeast studies. This work was supported in part by the Baylor College of Medicine MRRC (grant HD24064) and NIH grants GM52982, HD29256, and HD38038 to D.L.N.

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Article

RECEIVED FOR PUBLICATION JUNE 11; ACCEPTED AUGUST 9, 2002.

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