Human Molecular Genetics, 2000, Vol. 9, No. 3 439-445
© 2000 Oxford University Press
Dramatic, expansion-biased, age-dependent, tissue-specific somatic mosaicism in a transgenic mouse model of triplet repeat instability
Division of Molecular Genetics, Institute for Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, UK and 1Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Texas Medical Center, Houston, TX 77030, USA
Received 19 October 1999; Revised and Accepted 7 December 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Myotonic dystrophy type 1 (DM1) is one of a growing number of inherited human diseases whose molecular basis has been implicated as the expansion of a trinucleotide DNA repeat. Expanded disease-associated alleles of >50 CTG repeats are unstable in both the germline and soma. Expansion of the unstable alleles over time and variation of the level of mutation between the somatic tissues of an individual are thought to account at least partially for the tissue specificity and progressive nature of the symptoms. We previously generated a number of transgenic mouse lines containing a large expanded CTG repeat tract that replicated a number of the features of unstable DNA in humans, including frequent sex-specific changes in allele length during intergenerational transmission. Small length change mutations were apparent in the somatic tissues of young mice in all of the lines generated, but the gross instability observed in human DM1 patients was not replicated. We now show that in one of the lines, Dmt-D, spectacular, expansion-biased, tissue-specific instability is observed in older mice. The highest levels of instability were detected in kidney with gains of >500 repeats, representing a tripling of allele length, in some cells. Mosaicism accumulated in an age-dependent manner, but the tissue specificity did not obviously correlate with cell turnover. Such gross somatic mosaicism was not observed in three other lines examined, further emphasizing a role for flanking DNA in modulating repeat stability.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The expansion of trinucleotide repeats has been identified as the mutational event underlying a growing number of inherited human diseases (1). The list includes the expansion of CGG repeats at the fragile X, A and E loci, and GAA repeats at the Friedreich ataxia locus. Most of the triplet repeat diseases, though, are associated with the expansion of CAG/CTG trinucleotides, including myotonic dystrophy type 1 (DM1), Huntington’s disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA) and the spinocerebellar ataxias (SCAs) types 1, 2, 3, 7 and 8 (1,2). In general, larger repeats are associated with a more severe form of the disease and an earlier age of onset. In all of these diseases the repeats are remarkably unstable and have been described as dynamic mutations (3). The repeats are particularly unstable in the germline and generally biased towards further expansion, accounting for the unusual genetics of these disorders. However, the repeats are also somatically unstable, frequently varying from tissue to tissue. It is likely that somatic mosaicism has some influence on the tissue specificity and progression of the symptoms in most of these disorders.
DM1, the most common adult-onset muscular dystrophy, is associated with the expansion of a CTG repeat located in the 3'-untranslated region (3'-UTR) of the DM1 protein kinase (DM1PK) gene (4–6) and the promoter of the SIX5 gene (7–9). The CTG expansion in DM1 shows the widest range of expansions yet reported, with disease alleles ranging in length from 50 up to many thousands of repeats. The repeat is very unstable in the germline and biased towards large expansions, accounting for the very high levels of anticipation characteristic of DM1 (10). Somatic mosaicism in DM1 is also very high. Specifically, DM1 demonstrates age-dependent (11), expansion-biased (12) mosaicism that appears to be mediated via multiple small length change events (13). Most interestingly, in older individuals the average allele length in muscle, the primary affected tissue in DM1, is always larger than that observed in circulating lymphocytes (14–16). The relative stability of the repeats in additional tissues in DM1 individuals is less clear, with far fewer individuals having been analysed. General findings suggest that repeat expansions in blood are the smallest and, in addition to muscle, heart and kidney, usually display the largest expansions (17–23). Thus, the average allele length measured in DM1 individuals depends on both the age of the individual and the tissue sampled. Failure to correct for the age-at-sampling effect has almost certainly compromised attempts to correlate repeat length and disease severity, and interpret intergenerational transmissions fully (13). Moreover, since it is known that larger repeats are associated with a more severe form of the disease, it would seem logical to assume that age-dependent, expansion-biased, tissue-specific somatic mosaicism contributes at least in part to the tissue specificity and progressive nature of the symptoms.
Neither the molecular mechanisms underlying repeat instability nor the basis of variability between tissues at any of the expanded repeat loci are understood. Further analysis of these processes in patients is limited by the availability of appropriate samples throughout the lifetime of an individual and is further compromised by inter-individual genetic and environmental variation. To facilitate more detailed studies of repeat dynamics and the mutational mechanisms underlying instability throughout mammalian development, a number of murine models of CAG/CTG repeat diseases and/or expanded repeats have been developed. These include models for a number of loci and with varying amounts of flanking DNA in simple transgenics (24–32), and more recently recombination into the murine homologue (33,34). Not all of these models exhibit detectable inter- generational length changes (24–26,32) and in those that do the length changes observed do not replicate the magnitude of those observed at human loci such as DM1 (27–31,33,34). Similarly, not all of these models exhibit detectable somatic mosaicism (24–26,30,32). In those lines that do show detectable somatic mosaicism, there is little evidence for instability in tissues in young mice, but in older mice clear differences are observed between tissues (28,31,33,35). Although somatic instability in all the mouse models is expansion biased, the length changes observed are relatively small, typically <20 repeats. This limited level of somatic instability has led to the suggestion that fundamental differences exist in the metabolism of expanded repeat sequences between mice and humans, and that the very high levels of somatic mosaicism observed at the DM1 locus in humans, with length changes of 100 repeats or more, may not be reproducible in the mouse. We now demonstrate that this is not the case.
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RESULTS |
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Somatic mosaicism in old Dmt162 transgenic mice
We have previously generated and performed a preliminary characterization of four mouse lines containing the Dmt162 transgene (27). The transgene construct comprised ~162 CTG repeats and ~750 bp of flanking DNA from the human DM1 locus, including most of the DM1PK 3'-UTR. It included no coding DNA or promoter elements and was intended only to model expanded repeat instability. We had previously established that although frequent small length changes were observed in the tissues of young mice, the gross tissue-specific, expansion-biased instability seen in DM1 patients was not replicated (27). To determine whether greater levels of instability might be observed in older mice, we harvested the tissues from a 20-month-old male and female mouse from each of the lines Dmt-B, -C, -D and -E. Replicate small pool polymerase chain reactions (SP-PCRs) (13,36) containing ~10–100 molecules from each of 12 tissues (spleen, lung, liver, heart, kidney, brain, colon, diaphragm, eye, tongue, skeletal muscle and tail) were performed and compared with tail DNA isolated shortly after weaning (~6 weeks old) (Fig. 1). As with the young mice characterized previously, the level of mosaicism in the three lines Dmt-B, -C and -E did not reveal significant somatic mosaicism; length change mutants over ±20 repeats were not detected in any of the tissues tested in either sex. In the Dmt-D line, however, there was evidence for significant instability in a number of tissues in both the male and female mice. Most notably, the repeat was dramatically unstable in kidney with a clear bias towards large expanded alleles. Indeed, a subset of cells from the kidney of 20-month-old mice contained alleles in excess of 650 repeats, representing gains from the progenitor of >500 repeats. Other tissues in which the repeats were particularly unstable included liver, colon, skeletal muscle, diaphragm and eye. Tissues in which the repeat remained relatively stable were tail, spleen, heart and lung. Nevertheless, even in these tissues, there was an increase in the average allele size detected from that observed in the tail DNA at weaning.
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Age dependence of somatic mosaicism in Dmt-D mice
To delineate further the time at which somatic mosaicism accumulates in Dmt-D mice, DNA was extracted and analysed from mice at 2, 6 and 13 months of age (Fig. 2). At 2 months of age, the repeat appeared relatively stable in most of the tissues, but with the tissues in which the repeat is most unstable, such as kidney and colon, starting to accumulate a fraction of cells with relatively large (>20 repeats) length change variants. By 6 months of age, mosaicism was apparent in all tissues tested and continued to accumulate with age, indicating that new mutations continue to occur throughout the lifetime of the mouse. The relative stability of the repeat within the tissues appeared to be consistent between the mice tested at different age points. To investigate further the reproducibility of the tissue specificity of the instability observed, three male and three female mice were harvested at 12–13 months of age. Once again the relative stability of the repeat within the tissues appeared to be preserved, with kidney cells in both sexes consistently showing the greatest levels of repeat variation and containing the largest expansions (data not shown).
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Single-molecule analyses of somatic mosaicism in the lung and kidney of Dmt-D mice
In a more detailed analysis of the progression of variability in the lung and kidney of Dmt-D mice, the process was quantified by SP-PCR at the single-molecule level. For each tissue at each time point (2, 6, 13 and 20 months), >100 single molecules were amplified and sized (Figs 3 and 4). Since major length change variants are not observed in the tissues of young mice, we have assumed that the allele size measured in the tail DNA of very young mice represents the progenitor allele. In the kidney DNA from a 2-month-old mouse, >30% of the cells have acquired increases in allele length of >10 repeats. None the less, a detectable fraction of cells, ~7%, have acquired relatively large contractions, >5 repeats. These data indicate that although there is a clear overall bias towards gains in repeat length, at least a fraction of events result in loss of repeats. At 13 months of age, mosaicism in the kidney is very dramatic with some cells containing expansions of >200 repeats relative to the progenitor. By this stage, it is also apparent that variability within the kidney is not homogeneous. Rather, it would appear that the repeat is not equally liable to mutation in all cells, and that three major subpopulations of cells may have differing mutational dynamics. The cells in which the repeat is most stable, ~35% of the total population, had attained a mean allele size ~7 repeats larger than the progenitor (peak I). The cells with the intermediate dynamics, ~40% of the total population, had acquired mean gains of ~50 repeats (peak II), whereas the population of cells (~25% of cells) in which the repeat is the most unstable had acquired a mean allele length of +150 repeats (peak III). By 20 months, the trimodality was even more apparent with peaks of approximately +10, +100 and +270 repeats. To confirm the multimodality of DNA length variants observed in kidney cells, we performed additional SP-PCR reactions with ~200 cellular equivalents of DNA per reaction from a 13-month-old mouse (Fig. 5). Although with such high input DNA concentrations individual alleles could no longer be resolved, the multimodality in the distribution of variants was clear. Similar multimodality was observed in the kidney of all of the older Dmt-D mice tested (>12 months, n = 8). In contrast, multimodality was not observed within the lung cell populations, although there was a gradual increase in allele length, including a tail of larger alleles.
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Regional mosaicism in the brains of Dmt-D mice
Since most of the CAG/CTG repeat expansion disorders are neurodegenerative diseases, much emphasis has been placed on examination of repeat mosaicism in brain. Microdissection of brain regions from a 12-month-old Dmt-D mouse revealed clear differences in the relative levels of variability (Fig. 6). Relatively high levels of variation were observed in most regions, in particular in the hindbrain and striatum, which showed a mean increase of ~25 repeats relative to that observed in weaned tail DNA. The cerebellum was clearly the most stable region of the brain, although even in this area the average allele size had increased by ~12 repeats relative to that observed in weaned tail DNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have demonstrated that the Dmt-D line of transgenic mice replicates the gross somatic mosaicism observed in DM1 patients. Most notably, mosaicism accumulates in an age-dependent manner, at a tissue-specific rate and is expansion biased with especially large length change events. In some cells of older mice, gains of >500 repeats, indicating a tripling of the progenitor allele size, have been observed. The detected levels of mosaicism are at least comparable to, if not greater than, that observed in blood DNA of DM1 individuals with similar sized alleles (e.g. see DM-4 and DM-5 in ref. 13). Although DM1 is a multisystemic disorder, skeletal muscle weakness is a hallmark of the disease. In older DM1 individuals, the average allele length in muscle is always larger than that observed in circulating lymphocytes (14–16). Similarly, the repeats in the main skeletal muscles analysed in this study, the quadriceps femoris, and in the diaphragm and tongue, were much more unstable than in leukocyte populations (as revealed by spleen DNA analysis), although not obviously different from each other. Difficulty in obtaining samples from an extensive range of somatic tissues from DM1 individuals has resulted in a lack of detailed somatic stability data from humans. Thus, a precise tissue-by-tissue comparison cannot be made. None the less, when comparing the oldest mice assayed with the human samples available, it would appear that the level of repeat instability observed in the Dmt-D mice is at least as great as that observed in DM1 individuals. In terms of the rate of change as a function of time, it may even be greater. These data refute suggestions that fundamental differences in DNA triplet repeat metabolism between mouse and man represent an insurmountable block to replicating gross instability in murine models.
Such dramatic somatic mosaicism involving large length change mutations has not been observed previously in numerous transgenic models of triplet repeat instability (24–26,28–35). Nevertheless, age-dependent, tissue-specific, expansion-biased somatic mosaicism has been observed in a number of these models (28,31,33,35). The failure to detect such large length changes in these other models may simply reflect the smaller array size integrated, or it may represent a failure to transfer and/or integrate next to appropriate cis-acting sequence modifiers (see below). Alternatively, some of these models may actually have more dramatic instability than has been recognized so far. Our own studies have revealed that the electrophoretic profiles generated by bulk PCR analysis with fluorescently labelled primers and detection on automated DNA sequencing type apparatus [as has been used to characterize mosaicism in some of the transgenic models (28,31,33)] greatly underestimates the true level of variability within a sample, in particular the presence of large expanded alleles. For instance, our sensitive SP-PCR procedures have revealed expansions in the kidney DNA of Dmt-D mice up to 600 repeats in length, whereas the electrophoretic profiles of the same DNA reveal only limited mosaicism with no detectable signals from alleles >200 repeats in length (data not shown).
The pattern of tissue specificity of mosaicism revealed in the other repeat transgenic models appears similar to that of Dmt-D. Most notably, kidney, liver and striatum appear particularly unstable, and cerebellum and spleen particularly stable, in all of the lines. The relative stability of the repeats in the cerebellum is especially noteworthy. Not only are repeats in the cerebellum very stable in mice, but also this tissue shows only low levels of mosaicism in all of the human samples analysed with expanded repeats, including brains from individuals with HD (37), SCA1 (38), SCA3 (39), DRPLA (40) and DM1 (41). The apparent conservation of repeat stability profiles between the various transgenic lines and humans suggests that the genomic context of the repeat may have little effect on the relative stability observed between different tissues. Rather, they suggest that the major factors influencing the tissue specificity of repeat instability are mediated via tissue-specific trans-acting genetic modifiers. This would appear to preclude a direct role for gene transcription in promoting instability, as tissue specificity of repeat instability would be expected to parallel promoter activity in a gene-dependent manner. Indeed, Lia et al. (35) have demonstrated no association of somatic mosaicism with DM1PK transcriptional activity in their DM1 region repeat transgenic mice (35). The Dmt162 transgene contains no promoter elements and transcription would only be expected if it fortuitously integrated within a gene. Conversely, Mangiarini et al. (28) have demonstrated an association in different transgenic lines between transgene expression and relative stability, as their most stable line is not expressed. However, this could reflect a requirement for an open chromatin structure, rather than for transcription per se (42). Similarly, levels of instability do not appear to correlate with cell division rates (43), as also noted previously (35). In particular, the tissue in which the repeat is the most unstable reported here, kidney, has a modest rate of cell turnover, comparable to that of lung (43), a tissue in which the repeat is clearly much more stable. As in DM1 patients (14–16), the rates of expansion are also greater in slowly proliferative muscle cells than in more rapidly dividing leukocyte populations. Similarly, prominent mosaicism in DM1 newborns (11) and in the transgenic mouse models is not observed, despite the rapid rate of cell division that occurs during embryogenesis. Moreover, there are high levels of mosaicism in many brain regions of older mice, despite the fact that neuronal populations in the adult are post-mitotic. It should of course be considered that the majority of cells in the brain are supporting glia, rather than neurons. Even so, glia divide less than once in normal mice >4 months old (44,45). Yet, even in the most stable regions of the brain, such as the cerebellum, the vast majority of the cells of older mice contain expansions relative to the allele observed in the tail DNA of the young mice. Combined, these data argue against a direct link between cell division and the mutation process. They do not preclude such a link, but indicate that other factors providing tissue specificity must also be of critical importance. More detailed studies will be required to ascertain whether mutations can accumulate in non-dividing cells, as suggested by these data.
In addition to differing levels of mosaicism between tissues, we have also revealed clear intra-tissue differences, not only in brain as has been previously observed, but also within kidney. Detailed analysis of repeat variability in the kidney of Dmt-D mice revealed at least three distinct cell populations with differing repeat dynamics. These data indicate that repeat instability is, not surprisingly, cell type specific as well as tissue specific. The ability to detect cell type-specific variation within a tissue depends on the relative level of variation between cell types, the relative proportions of cell types within a tissue and the sensitivity of the detection methods employed. It seems likely that intra-tissue variation in cell type-specific mutational dynamics is even more common than we have reported here. Small subsets of cells with much larger alleles than the bulk of the tissue population may be missed unless very sensitive techniques are used. Such cell type-specific mutational dynamics might have important implications for some of the repeat expansion disorders, in particular in those diseases in which only specific subpopulations of cells are vulnerable. We currently do not know which specific cell types are the most unstable in the tissues we have examined. The kidney, in common with tissues in which the repeat remains relatively stable, has nervous, lymph and vascular systems, and we can thus speculate that these cell types are not the origin of the large kidney-specific expansions. We hypothesize, therefore, that the dramatic instability detected in the kidney is accounted for by one or more highly specialized nephron cell types, which are unique to the kidney. Future use of microdissection and/or cell sorting procedures will be utilized to address this issue in more detail.
Clearly, the repeat instability observed is expansion biased over time. Yet, deletion mutants are observed in a subset of cells, in particular in young mice, suggesting that the mutational process is bi-directional, but that an overall expansion bias results in the majority of cells acquiring expansions relative to the progenitor allele over time. Whether this expansion bias represents larger and/or more frequent gains relative to losses remains to be determined. Similar arguments can be made regarding the relative levels of variation observed between tissues; these could also result from differences in absolute mutation rates and/or differences in mutational length change sizes. The size of most length change events appears to be quite small. For instance, in the cerebellum there is a clear shift upwards in the mean allele size over time, although the overall level of variation within the tissue remains relatively low. Such a homogeneous shift in the cell population is consistent with more frequent small length changes rather than rarer large length changes. Similar patterns of repeat length changes are also observed in the tissues in which the repeat is more unstable, although the distributions are clearly skewed towards larger alleles. The tail of larger expanded alleles is consistent with a length-dependent increase in mutation rate and/or size of the length change events.
Although we observe gross somatic instability in these mice with large length changes that accumulate in an age-dependent manner, preliminary data indicate that germline instability in these mice remains relatively modest. Although the Dmt-D line is the most unstable of our lines (27), even from older mice, the intergenerational differences observed are still usually <10 repeats (data not shown). Moreover, despite the clear expansion bias observed in somatic tissues, mutations in the female germline stay biased towards deletions. Similar age-dependent increases in germline mutation rates have been observed for some of the other transgenic lines, although they too still only show modest repeat length changes (28,30,31,46). These data further highlight the tissue specificity of repeat metabolism and suggest that the clear differences observed between the somatic and germinal pathways in humans (13) may be replicated in the mouse. However, they also suggest that whatever the genomic context that facilitates gross somatic instability to be observed in the Dmt-D line, it does not extend to the facilitation of gross instability in the germline. Alternatively, it may be that germinal instability is critically dependent on time per se, in which case even the most unstable mouse locus may be not be able to replicate the large length changes of the more unstable loci observed in humans of reproductive age (42).
Gross somatic instability was observed only in Dmt-D. The three other simple Dmt162 lines, Dmt-B, -C and -E, exhibited only limited instability despite the transgene construct and array size being the same. The difference in their relative stability must therefore be due to position effects associated with the site of transgene integration. Such data further highlight the considerable effect that flanking DNA sequences have in modifying expanded repeat stability. We have recently revealed striking correlations between germline CAG/CTG repeat expandability and flanking GC content and proximity to CpG islands in humans (47). The flanking DNA incorporated within the transgene is derived from the human DM1 locus and has a high GC content (63%) and CpG observed over expected ratio (0.81), and qualifies as a CpG island (48). However, it is much shorter (742 bp) than the full-length island present in humans [~3 kb (7)] and is asymmetrically apportioned either side of the repeat (133 bp on the 5' side and 609 bp on the 3' side). Whether the transgene sequence displays the expected CpG island properties of hypomethylation remains to be determined. None the less, it would appear that this amount of flanking DNA is not able to mediate position-independent gross somatic mosaicism. Studies are ongoing to determine the site of integration of our transgenes and attempt to elucidate how flanking DNA sequence effects are mediated.
The Dmt-D line of transgenic mice should provide an excellent model system for further exploring the dynamics of the somatic expansion process, providing an unlimited source of material throughout all time points of mammalian development. Moreover, additional comparisons between this unstable line and the other more stable lines should provide further insights into the nature of the cis-acting sequence modifiers that clearly exert a profound influence on repeat stability.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse DNA and PCR
Mouse tissue DNAs were prepared as previously described (27). The skeletal muscle used was from the quadriceps femoris. All mice were hemizygous for the transgene on a pure FVB/N background strain and had estimated progenitor allele lengths of between 150 and 180 CTG repeats. PCR was performed as by Monckton et al. (27) in a Biometra UNO thermoblock. SP-PCR was carried out as described previously (13) with 0.1 µM of carrier primer DM-H, and using PCR primers DM-H (27) and DM-DR (13).
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ACKNOWLEDGEMENTS |
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We would like to thank the University of Glasgow Dynamic Mutation Group for helpful discussions with this work, in particular we would like to thank Heather Johnston for excellent assistance with microdissection. We are also indebted to the staff of the Biological Services for excellent animal care and assistance with colony maintenance and tissue harvesting. D.G.M. is a Lister Institute Research Fellow. This work was made possible by awards to D.G.M. from the Medical Research Council (UK) and to M.J.S. from the MDA and Mr Kenneth D. Muller.
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FOOTNOTES |
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+ Present address: 9 Aristotelous Street, 15234 Halandri, Athens, Greece
§ To whom correspondence should be addressed. Tel: +44 141 330 6213; Fax: +44 141 330 6871; Email: dmonck@molgen.gla.ac.uk
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REFERENCES |
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