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Somatic instability of the CTG repeat in mice transgenic for the myotonic dystrophy region is age dependent but not correlated to the relative intertissue transcription levels and proliferative capacities
Introduction
Results
The CTG repeat mosaicism is tissue specific and larger in older mice
Detailed SP-PCR analysis of the somatic instability
Relationship between transcription and instability
Discussion
Materials And Methods
CTG repeat analysis
SP-PCR analysis
Semi-quantitative RT-PCR
Acknowledgements
References
Somatic instability of the CTG repeat in mice transgenic for the myotonic dystrophy region is age dependent but not correlated to the relative intertissue transcription levels and proliferative capacities
INTRODUCTION
Myotonic dystrophy (DM) is a dominant autosomal multisystemic disorder involving myotonia, progressive weakness and wasting of muscle and extramuscular symptoms such as cataracts, cardiac conduction defects, testicular atrophy and endocrine and cognitive dysfunction (1). DM is caused by expansion of a (CTG)n trinucleotide repeat in the 3[prime]-untranslated region (UTR) of the DM protein kinase gene (DMPK) on chromosome 19 (2-5). In normal individuals, the (CTG)n repeat is polymorphic and varies between five and 37 (3-6), whereas there are 50-4000 CTGs in affected patients (7,8). The size of the (CTG)n repeat, which increases from generation to generation, is generally correlated with clinical severity and age at onset, providing a molecular basis for the anticipation phenomenon observed in DM families (9). The instability of the CTG repeat from generation to generation depends on its size and the sex of the transmitting parent (10-14). Somatic instability, resulting in broad smears on Southern blots, is observed in various tissues of DM patients reflecting length mosaicism between cell lineages (8,11,15-19). Expansion of the CTG repeat in blood cells is continuous throughout the life of DM patients, the rate depending mainly on the initial size of the expanded allele and time (20). The expansion process may also be affected by unknown individual-specific factors.
We do not know when trinucleotide expansion occurs, and the molecular mechanisms of the instability remain elusive. Recreating trinucleotide repeat instability in mice could thus provide a useful tool for studying the mechanisms involved. The repeat stability initially observed in mice transgenic for a CAG repeat suggested that the molecular mechanisms underlying triplet repeat instability in humans may not exist in mice (21-23). However, we and others demonstrated intergenerational and somatic cell instability of a trinucleotide repeat in mice (24-26). Interestingly, Mangiarini et al. (26) found that the only stable CAG repeat transgene was that present in the only line in which it was transcriptionally silent, suggesting that instability may be associated with the transcriptional activity of the transgene. Kaytor et al. also detected intergenerational CAG repeat instability in transgenic mice, but only when the transgene was maternally transmitted (27). They also demonstrated that maternal instability of the repeat occurs after meiotic DNA replication and before fertilization.
We investigated CTG repeat expansion mechanisms using mice transgenic for a 45 kb genomic fragment containing the 59, DMPK and DMAHP genes and 55 CTG (24). The fragment used for injection was cloned directly from a DM patient with cataracts. We analysed the CTG repeat length by small-pool PCR (SP-PCR) in various tissues to investigate the factors involved in the mechanisms underlying triplet instability, including tissue, age and level of expression of the transgenes.
RESULTS
The CTG repeat mosaicism is tissue specific and larger in older mice
We investigated the somatic instability of expanded (CTG)n repeats in tissues from 55 CTG mice of various ages from two lines carrying one copy each of the 45 kb human genomic DNA fragment. In a previous study, the DM55/85 line showed intergenerational instability with, by now, a mutation rate of 4.3%, and we also observed somatic instability in this line (24). The other line studied was DM55/86 which has not yet shown any intergenerational instability (over 33 descendants obtained) (24).
In this study, we investigated various tissues from 10 hemizygous mice from both sexes aged between 4 and 19 months by PCR with primers 101/102 and 6% polyacrylamide gel as previously described (24). We detected, in both lines, various bands containing >55 CTG repeats, reflecting mosaicism and somatic instability (Fig.
Figure 1. Somatic instability in transgenic mice of various ages. PCR analysis of CTG repeats in various tissues from two lines carrying one copy of the 45 kb genomic human fragment (lines 85 and 86). (A) Lung, kidney, pancreas, liver, ovary, eye, quadriceps, gastrocnemius and heart. (B) Cerebellum, thalamus, cerebral hemisphere and olfactory bulb were tested for line 86. (C) Leucocytes were tested for lines 85 and 86. The fragment injected was also amplified to check the size of the original 55 CTG repeat (c). We also examined the instability of the CTG repeat in various parts of the brain and in leucocytes from DM55/86 and DM55/85 mice (Fig. 
Detailed SP-PCR analysis of the somatic instability
We analysed in detail the extent and dynamics of amplification, using the SP-PCR procedure described by Monckton et al. (17). We examined six tissues from DM55/86 mice (Fig.
Figure 2. Schematic representation of the SP-PCR analysis of various tissues at two ages. The x-axis is the length of the CTG repeat and the y-axis is the percentage of cells containing n CTGs. Results for six tissues from two mice (line 86) of different ages are displayed: heart, gastrocnemius, eye, liver, pancreas and kidney. On the left, from a 4-month-old mouse, and on the right, a 19-month-old mouse. Six picograms of BamHI-digested DNA was used for SP-PCR experiments. In a 4-month-old mouse, most cells in all tissues tested contained 55 CTG repeats. However, there was mosaicism in all the tissues at this age, with cells containing from 47 to 61 CTG repeats. Kidney, liver and pancreas had a larger variety of CTG repeat lengths [only 47% (kidney), 56% (liver) and 51% (pancreas) of cells with 55 CTG] than heart (74%), muscle (68%) and eye (63%). The percentage of cells containing 55 CTG repeats was lower in a 19-month-old mouse, and the distribution of the number of CTG repeats (45 up to 67) was more spread out than at 4 months. This was particularly evident in the kidney, pancreas and liver, confirming that there was a higher level of somatic mosaicism of the CTG repeat in these tissues. We also looked at the distribution of CTG repeats in various tissues (Table 1). At 4 months of age, in all tissues except the eye, there was a bias towards expansion of the CTG repeat which was greater (about twice) in the liver, pancreas and kidney than in the heart and muscle. At 19 months of age, the tendency for the repeat to expand was higher, showing that the rate of expansion was greater than the rate of contraction, and differed between tissues. SP-PCR with DNA from the heart and liver of DM55/85 mice also showed the same bias towards expansion of the CTG repeat (not shown).
Table 1.
| 4-month-old mouse | 19-month-old mouse | |||||
| <55 | 55 | >55 | <55 | 55 | >55 | |
| Heart | 13.75 | 66.25 | 20 | 15 | 53.75 | 31.25 |
| Gastrocnemius | 12.25 | 68.75 | 18.75 | 20 | 52.5 | 26.25 |
| Eye | 23.75 | 61.25 | 16.25 | 27.5 | 30 | 42.5 |
| Liver | 8.75 | 55 | 36.5 | 22.5 | 25 | 52.5 |
| Pancreas | 12.5 | 55 | 33 | 25 | 21.5 | 55 |
| Kidney | 20 | 50 | 30 | 28 | 31.25 | 41 |
Relationship between transcription and instability
Analyses of mice transgenic for exon 1 of the huntingtin gene carrying CAG expansions showed that there was intergenerational and somatic instability in all lines except the line that did not express the transgene. This suggests that instability may be linked to gene expression as a consequence of the open chromatin structure, which may favour triplet repeat amplification, or by a direct link to transcription (26).
Expression of the three human genes (59, DMPK and DMAHP) in our transgenic mice was assessed by reverse transcription (RT)-PCR in brain, heart, muscle and kidney tissues from each of the lines, using the mouse TBP (TATA-binding protein) gene as an internal control (29) (Fig.
Figure 3. RT-PCR analysis of the expression of the integrated human DMPK, DMAHP and 59 genes in various tissues: gastrocnemius, heart, brain and kidney. A representative analysis of expression of the three genes is shown in a non-transgenic control (C), and in a transgenic animal from the single copy line 85 (T). RNA from the transgenic animal was tested with RT (+) and without RT (-) to check for contamination by genomic DNA. RT-PCR analyses were performed with two sets of primers (see Materials and Methods) specific for the tested gene (lower band) and for the internal control, the mouse TBP (upper band). We investigated whether the level of expression was correlated with the level of somatic instability using semi-quantitative RT-PCR to determine the level of expression of the 59, DMPK and DMAHP genes in the tissues assessed for somatic instability (Table 2). As the levels of expression of these genes did not seem to vary very much in adult mice, we can report that we did not observe any correlation between the level of CTG repeat somatic instability and the level of mRNA of the three neighbouring genes. For example, the tissues with the highest level of mosaicism (liver or kidney) were not necessarily those with the highest level of expression of any of the three genes (Table 2). Table 2. 
Gene
Age (months)
Heart
Gastrocnemius
Liver
Kidney
DMPK
4
7.8
1.8
0.45
0.4
19
8.7
1.6
0.98
0.69
DMAHP
4
1.3
0.67
1.84
0.74
19
1.58
0.23
2.36
0.75
59
4
0.5
0.72
0.37
0.63
19
0.57
0.2
0.47
0.68
DISCUSSION
In this study, we have demonstrated the somatic instability of a 55 CTG repeat carried by a 45 kb human DM genomic DNA fragment in transgenic mice. In six out of seven lines, we observed an intergenerational instability. This instability was moderate, probably due to the small repeat size. Considering all these six lines together, we did not observe any parental bias in the frequency or in the length of the size variation (24), bias which has been observed in other transgenic models carrying unstable CAG or CTG repeats (25-27). However, as reported in DM families, in our mice there was a significant bias towards expansion of the repeat (only 5% contraction in mice). The large human genomic sequence used to generate our transgenic mice may favour trinucleotide expansion. Surrounding sequences and the chromatin environment are likely to be involved in trinucleotide repeat instability. All seven of our lines expressed the transgenes in a tissue-specific manner (at least for 59 and DMPK), suggesting that the appropriate chromatin structure is recreated in transgenic mice regardless of the DNA integration site. It also implies that the major transcriptional regulatory elements were included in our constructs.
Only one of the two one-copy lines analysed herein (line DM55/85) was moderately unstable between generations, with a mutation rate of 4.3%. The other line, DM55/86, has not yet shown any intergenerational instability in the 33 descendents studied, but has a level of somatic instability similar to that of the DM55/85 line. CTG repeats are also unstable in this line, and intergenerational instability might be detected with a larger progeny. In both lines, as for DM patients (16,18,30,31), there was CTG repeat somatic instability throughout adult life. Therefore, transgenic mice carrying a 55 CTG expansion in its human chromosomal context appear to be a convenient model for studying instability mechanisms and its progression over time in diverse tissues. Somatic instability was also observed throughout adulthood for the CAG repeat in mice transgenic for the first intron of the huntingtin gene carrying ~150 CAG repeats.
We only observed somatic instability in adult animals. In young animals, all tissues generated a major band consisting of 55 CTG repeats, this being the size of the inherited CTG repeat, with very few other bands produced by somatic instability barely detectable. In DM patients, as already suggested by Jansen et al. (14), it seems that two types of somatic instability can be defined, which may be due to different mechanisms: a first wave of pronounced instability occurs very early in embryogenesis, probably between 13 and 16 weeks into gestation leading to major differences between tissues (32). The degree of heterogeneity with the original expansion size does not appear to reflect the number of cell divisions during development or within lineage. The second wave of instability, which is more reduced, could follow the first wave and continue throughout adulthood. This results in a `smear' on Southern blots (8,11,15-19). We did not observe the first wave of instability in our transgenic mice with 55 CTG repeats, suggesting that the size of the repeat may be limiting or that the mechanisms involved in humans, during development, do not occur in mice.
Somatic instability occurred in adult DM55 mice at different rates in different tissues. The same tissues in the two lines studied, the liver, pancreas and kidney, had the highest level of mosaicism. There was also a high level of mosaicism in brain tissues. There was less expansion of the CTG repeat in the cerebellum than in other regions of brain. This has also been shown for CTG expansions in DM patients (28) and for the CAG repeat in Huntington's disease (33), spinocerebellar ataxia types 1 and type 3 (34,35) and dentaturobral-pallidoluysian atrophy (36) patients. SP-PCR showed that the instability is biased towards expansion as observed in DM patients (17) and that the dynamics of repeat length changes differ between tissues. Why does instability differ between tissues? Trinucleotide repeats instability studies in vitro (19,37), in Escherichia coli (38,39) and in yeast (40-44) have clearly shown that replication and DNA mistmatch repair are involved in trinucleotide instability. If instability results only from mitotic events, there should be a correlation between the rate of length changes and proliferative capacity. Messier and Leblond used [3H]thymidine to study cell proliferation in various mice and rat tissues. They found that liver, pancreas and kidney tissues, which had the highest level of repeat instability in our study, grow and divide more than heart and muscle tissues (45). However, this explanation did not fit all the tissues we examined. Various parts of the brain had significant levels of instability although neurons do not divide. Nevertheless, other cell types, such as glial cells, may be responsible for the observed instability. Repeats in renewing lung cells and leucocytes are stable in our mice, suggesting that if instability occurs during cell division, then DNA repair systems, which may be involved in instability mechanisms, must be more efficient in these tissues. Alternatively, instability may occur independently of replication in non-dividing cells. Such a mechanism may account for instability occurring in mice oocytes with advancing maternal age (27). The repeat may adopt particular structures, such as a hairpin loop (46,47), which may be recognized by DNA repair systems in quiescent cells. The repair machinery involved may slip on the repeat, increasing the repeat length. This would make expansion possible in non-dividing tissues and the differences between tissues would be accounted for by differences in DNA repair efficiency, either in recognizing the CTG structure or in the repair itself. The human homologue of the E.coli MutS protein, MSH2, which binds (CTG)n(CAG)n slipped structures with an affinity that increases with repeat length, could be involved in these mechanisms (48). It has also been suggested that trinucleotide instability may be associated with gene expression, due to the open chromatin structure, which may favour triplet repeat mutation, or that it may be generated by transcription itself. We found no correlation between the level of somatic CTG repeat instability and the level of transcripts of the human genes in transgenic mice. Although differences in mRNA turnover have not been measured between tissues and cannot be excluded, it seems unlikely that it could explain the lack of correlation between somatic mutation rates and transcription levels. Thus, if expression and open chromatin facilitating unusual repeat structures are required for instability, the levels of transcription of the 59, DMPK and DMAHP genes in 4- and 19-month-old mice cannot account for the differences in the level of somatic mosaicism between tissues. This implies that, as has also been observed in E.coli (49), different pathways are involved in the CTG repeat instability in transgenic mice and probably in DM patients. Besides instability mechanisms linked to replication or transcription, others mechanisms independent of the replication and transcription machineries could also occur. Further analysis is required to define the nature of those mechanisms more precisely.
MATERIALS AND METHODS
CTG repeat analysis
PCR analyses were performed in a volume of 100 µl, containing 16.6 mM (NH4)2SO4, 67 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 67 mM EDTA, 10% dimethylsulfoxide (DMSO), 10 mM 2-mercaptoethanol, 0.2 mM dNTPs, 0.2 µM of primers 101 and 102 (101: 5[prime]-CTTCCCAGGCCTGCAGTTTGCCCATC-3[prime]; 102: 5[prime]-GAACGGGGCTCGAAGGGTCTTGTAGC-3[prime]). PCR involved heating to 94°C for 6 min, 65°C for 1 min, 72°C for 1 min and then 30 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 1 min. Amplified product (2 µl) was mixed with 2 µl of formamide loading buffer, heated for 5 min at 100°C, subjected to electrophoresis in a 6% denaturing acrylamide gel, blotted on to nylon membrane and probed with the 3[prime]-end-labelled 101 primer.
SP-PCR analysis
DNA samples were digested with BamHI. The digested DNA (6 pg) was amplified in 7 µl reactions using a previously described buffer system (50), 1.4 µM primer 101, 1.4 µM 102 and 0.14 U/µl of AmpliTaq Gold DNA polymerase (Perkin Elmer-Cetus). The DNA was denatured by heating to 95°C for 10 min. Reactions involved 28 cycles of 94°C (45 s), 66°C (45 s) and 70°C (3 min) with a chase of 66°C (1 min) and 70°C (10 min) in a Perkin Elmer 9600 thermal cycler. Amplified product (3 µl) was mixed with 2 µl of formamide loading buffer, heated for 5 min at 100°C, subjected to electrophoresis in a 6% denaturing acrylamide gel at 55 W for 3.5 h, blotted onto nylon membrane and probed with the 3[prime]-end-labelled 101 primer. The hybridized bands were detected by autoradiography and their size determined by comparison with M13 sequence maker and the PCR (101-102) product from our cosmid (55 CTG).
Semi-quantitative RT-PCR
Total RNA was extracted from various tissues by RNAzol (Bioprobe). Total RNA (1 µl) was used for the RT reaction with Superscript II (Gibco BRL) and random hexamer primers. The reverse transcription reaction was stopped by heating and the reaction mix was diluted 1 in 5 with water. The RT product (2 µl) was then used for the PCR, with two sets of primers: one set for the gene studied (59, DMPK and DMAHP) and one set for the murine TBP gene (internal control), 20 pmol of each primer was added, in a total volume of 50 µl containing 20 mM Tris-acetate, pH 9.0, 10 mM ammonium sulfate, 75 mM potassium acetate, 0.05% Tween-20, 10% glycerol, 2 mM MgSO4 for gene 59 (1 mM MgSO4 for DMPK and DMAHP), 200 µM each of dATP, dCTP, dTTP and dGTP, and 50 µCi of [[alpha]32P]dCTP. The mixture was heated for 6 min at 94°C, then 1 U of Tfl DNA polymerase (Promega) was added. Amplification was performed in a Perkin Elmer-Cetus thermocycler with denaturation at 94°C (1 min); annealing at 57°C (1 min) for gene 59 and 58°C (1 min) for DMPK and DMAHP; extension at 72°C (1 min); and 24 cycles (the optimum number of cycles, for which PCR was linear, with no heteroduplex formation). The labelled PCR products were subjected to electrophoresis in a 10% acrylamide gel. PCR products were quantified by PhosphorImager scans (Molecular Dynamics) using the IMAGEQUANT version 1.1 image analysis program (Molecular Dynamics). The peak area of the gene studied divided by the peak area for the murine TBP was used as the ratio for each sample. Primer sequences for RT-PCR reactions were as follows: DMPK, 5[prime]-GGACGACTTCGAGATTCTGA-3[prime] and 5[prime]-GCATGTCCCACTTGTTCATG-3[prime]; DMAHP, 5[prime]-TCATCAACTCCGGGGTGGGC-3[prime] and 5[prime]-CGGCCACACCCGTCACGATG-3[prime]; 59, 5[prime]-TCAAGTGCAGTACCTGGAT-3[prime] and 5[prime]-GTGTGATGCCAGGAACAGG-3[prime]; murine TBP, 5[prime]-GGTGTGCACAGGAGCCAAGAGTG-3[prime] and 5[prime]-AGCTACTGAACTGCTGGTGGGTC-3[prime].
ACKNOWLEDGEMENTS
This work was supported by grants from INSERM, the Délégation à la Recherche Clinique, Assistance Publique-Hôpitaux de Paris, the Association Française contre les Myopathies (A.S.L., G.G.), the Leg Poix and Université René Descartes.
REFERENCES
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