Human Molecular Genetics, 2000, Vol. 9, No. 18 2707-2713
© 2000 Oxford University Press
Sequence interruptions confer differential stability at microsatellite alleles in mismatch repair-deficient cells
University of Edinburgh Department of Oncology and MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Received 24 July 2000; Revised and Accepted 13 September 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Determinants of instability at a given microsatellite repeat merits investigation in view of relevance to understanding evolution of mutations at such sequences in human populations. The microsatellite D2S123 was studied as a paradigm CA repeat marker. Furthermore, this marker is one of a recommended panel used in molecular screening for hereditary non-polyposis colorectal cancer (HNPCC). In this investigation we show that the mutation rate at the D2S123 locus is markedly influenced by intra-allelic sequence variation within the repetitive tract itself. We employed a novel approach to characterize the nature of instability at D2S123, by utilizing cells derived from a non-tumour lineage, which harbour a dominant negative mismatch repair (MMR) mutation and a mutator phenotype. Individual alleles were typed using a semi-quantitative small pool PCR technique and this demonstrated substantial allele-these specific bias in susceptibility to mutation at the D2S123 locus. In support of these in vitro data, bias in allele mutation rate was also observed in tumours from 41 HNPCC patients, which was dependent on constitutional genotype. Sequencing of cell line and patient DNAs revealed that short alleles are significantly more susceptible to mutation due to the presence of uninterrupted CA repeats. Long D2S123 alleles are intrinsically more stable because of a TA interspersion within the repetitive tract. In addition to extending understanding of mutation at CA repeat dinucleotide tracts, these findings have considerable relevance both to screening programmes and to correlation of microsatellite instability (MSI) with colon cancer survival. The manifestation of tumour MSI may be substantially influenced by constitutional genotype.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Microsatellites occur ubiquitously throughout the genome and mutations within these simple repetitive tracts are frequent as evidenced by heterozygosity rates (1). In studies of poly(CA/GT) repeats in humans, high rates of mutation per locus per gamete per generation (5.6 x 10–4) have been revealed (2). The inherently unstable nature of microsatellites results in frequent alterations in the length of the repeat tracts making many of them highly polymorphic (1,3). Differences in mutation rate are evident at alternative microsatellite loci (1) and sequence variation can significantly affect individual microsatellite stability. Variations in microsatellite flanking sequences (4,5) and in repeat length (6,7) have been demonstrated to contribute to heterogeneity in mutation rate. In addition, correlation of instability with the degree of perfection of a repeat has been documented. In Escherichia coli and yeast, presence of an interspersion within a dinucleotide tract results in its stabilization (5,8); this is also observed in the presence of defective mismatch repair (MMR) (8). Locus-by-locus analyses reveal that interspersed repeat markers are also relatively more stable in the fruit fly Drosophila (9), in the germline of human populations (10) and in tumours from cancer patients (11). Repeat sequence interruption is also important at a clinical level. Expansion of triplet repeats gives rise to the human trinucleotide repeat disorders such as fragile X, Huntington’s disease and spinocerebellar ataxia type 1 (SCA1) (12,13). Stability of both SCA1 and FMR1 (fragile X gene) alleles are conferred by interruption of the contiguous repeat (14–18).
The high degree of polymorphism makes microsatellites invaluable tools for use in genetic mapping, in DNA forensic studies, as population markers and in a variety of other applications. Mechanisms affecting mutation rate within a given locus have been less extensively characterized, although there are studies documenting such variation. For example, in a population study of meioses in pentameric and tetrameric markers, it was shown that different alleles at single loci varied in their rate of mutation (10). However, investigation of mechanisms affecting microsatellite stability in family studies requires very large numbers of meiosis to be screened in order to identify mutations (2). This places severe practical limitations on such studies as well as on investigations using sperm DNA (19).
To investigate factors influencing stability at given microsatellite alleles we employed a novel approach using a ‘sensitized’ system. We utilized a lymphoblast cell line from a patient who developed colorectal cancer. The cell line is defective in MMR and displays a mutator phenotype but is derived from a non-tumourous tissue. There is evidence that mechanisms generating mutations in microsatellite unstable (MSI+) tumours share similarities with those resulting in the evolution of such sequences in the germline (11,20) and a mutator phenotype might exacerbate the process of germline evolution at repetitive markers. Hence, study of determinants of marker stability in MSI+ MMR-defective cells has relevance to the understanding of the process of evolution of repeat sequences in human populations (11,20).
Defects in mismatch repair result in the accelerated accumulation of frameshift mutations within microsatellite sequences (21–23) occurring predominantly at mononucleotide and dinucleotide repeat tracts. Over 90% of hereditary non-polyposis colorectal cancer (HNPCC) cases display microsatellite instability (MSI) (24,25), usually as a result of germline defects in one of at least five MMR genes (hMLH1, hMSH2, hPMS2, hPMS1 and hMSH6) (26,27). During tumour development, the wild-type allele is inactivated by loss of heterozygosity (LOH), mutation or epigenetic silencing causing complete loss of MMR activity. Some MSI+ sporadic colorectal tumours are also defective in MMR by nature of somatic mutations, LOH or by epigenetic silencing due to hypermethylation of the hMLH1 promoter (28). Once inactivated, the consequent failure to repair DNA replication errors that arise due to DNA polymerase slippage manifests as MSI.
In a cell line defective in MMR due to a dominant negative hPMS2 mutation, we show that alleles at a microsatellite repeat locus are differentially stabilized and demonstrate the sequence determinant of this mutation bias. The CA repeat microsatellite studied here is one of a panel of five markers recommended for use in MSI analysis in colorectal cancer (29,30). In addition to having general importance in understanding the mechanisms that lead to instability at repetitive sequences, this work also has specific relevance to the influence of patient genotype on the manifestation of tumour MSI and also on correlation of MSI with clinicopathological features.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Determination of allele bias in MMR-deficient cells
Genotyping of D2S123 alleles from MMR-defective lymphoblast cell line lbl-1261 revealed alleles of 228 and 214 bp (Fig. 1). In all, a total of 270 and 115 alleles were typed by small pool PCR (SP-PCR) for lbl-1261 and a control lymphoblast cell line (lbl-c5), respectively. Ninety alleles (33.3%) from lbl-1261 templates exhibited length variation compared with only two alleles (1.7%) of the control cell line (
2 = 42.3, P < 7 x 10–11) (Figs 1 and 2, Table 1). The small number of mutants found in lbl-c5 may be due to PCR error or indeed could be genuine mutations. However, their small number relative to those in lbl-1261 indicates that PCR artefacts do not interfere significantly with the detection of mutants in the dilute DNA. These data are in accordance with a previous study of mutation frequency in this cell line (31). However, we noted a substantial bias in the alleles from which mutations were derived. In all, 72 of 90 mutants (80%) in lbl-1261 were clustered around the shorter (214 bp) allele (Fig. 2A), indicating a substantial bias for mutation at that locus (2 = 42.3, P < 0.00001). Mutant alleles were assumed to be derived from the progenitor allele closest in size in the undiluted DNA, since studies of microsatellite mutations in human pedigrees, human cell lines and artificial constructs in yeast have shown that most microsatellite mutations involve only one or two repeat units (2,8,32).
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To ensure that the effect was not due to misassignment of mutant alleles, we re-analysed the data using a threshold to exclude reductions in the 228 bp allele. We assumed that only mutants of <216 bp (a single repeat expansion of the short progenitor allele) were derived from the shorter progenitor. This confirmed the highly significant mutation bias at the shorter allele (
2 = 8.45, P < 0.0037). These observations are not due to PCR bias at the smaller allele since equal numbers of short and long alleles were detected in lbl-1261. Furthermore, insertion and deletion mutants of both progenitor alleles were observed (Fig. 2). These observations indicate a substantial and statistically significant allele-specific bias in mutation rate. We were interested to determine the nature of this bias, especially since it conflicts with the expectation that MSI correlates with increased repeat length and number of repeating units (7,33)
Thirty-nine Scottish individuals were genotyped at the D2S123 locus. A total of eight different alleles were identified (210–230 bp) consistent with the CEPH data (http://www.cephb.fr/cephdb/ ) (Table 2). Alleles were noticed to cluster in two distinct size groups, long (~228 bp) or short (~214 bp) and the frequency of alleles in each of these two groups were almost identical between our cohort and the CEPH data.
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Mutation rate within the D2S123 CA repeat is predicted by sequence content
The sequence for D2S123 logged in GenBank is (CA)13(TA)(CA)15 (GenBank accession no. Z16551). Individual alleles were sequenced to determine whether sequence differences between long and short alleles might explain the substantial mutation bias. Initially undiluted lbl-1261 DNA was gel separated and individual alleles sequenced independently. This demonstrated that the 228 bp allele comprised of a (CA)28 repeat tract split into two runs of (CA)13 and (CA)15 by a TA dinucleotide, concurring with the sequence logged in GenBank. However, the shorter 214b bp allele comprised of an uninterrupted (CA)22 repeat with no interspersing TA dinucleotide (Fig. 3). Genotyping and sequencing of 15 D2S123 alleles from template DNA of a cohort of Scottish individuals showed that various length alleles classified as long always contained the TA interspersion, whereas various lengths of short alleles were invariably uninterrupted poly(CA) repeats. This analysis indicated that alleles clustered around 214 bp (short) contain perfect uninterrupted CA repeats. In contrast long alleles consistently contain a TA interspersion within the CA tract.
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In order to confirm definitively that mutant alleles arose predominantly from uninterrupted alleles in cell line lbl-1261, D2S123 alleles were PCR amplified from undiluted lbl-1261 template DNA and cloned. Individual alleles were sized and sequenced. Analysis of 28 clones confirmed that every long allele analysed contained the TA interruption and every short allele contained perfect CA repeats, thereby confirming that the presence of a TA interspersion confers stability on the CA repeats. Mutation was due to length variation of the number of core CA repeats in every case, implicating replication slippage as the causative mechanism of instability at this locus (Table 3). Cell line lbl-c5 was heterozygous for two short alleles and PCR cloning of undiluted DNA revealed two short uninterrupted alleles of (CA)21 and (CA)22 repeats. As expected in this MMR-proficient cell line, no mutations were detected in the clones despite both alleles having no interspersion of the repeat tract (data not shown).
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Taken together these data indicate that long alleles (
228 bp) invariably contain a TA interspersion in the repeat whereas short alleles (214 bp) contain uninterrupted (CA)n repeats that are inherently more unstable. This instability is unmasked in MMR-deficient cell lines.
Allele-specific bias of mutations at D2S123 in DNA MMR-deficient tumours
To determine whether allele bias might also influence MSI phenotype in MMR-deficient tumours, we genotyped D2S123 alleles in matched normal and tumour DNA samples from 41 colorectal cancer patients with MSI+ tumours. These have been characterized previously and exhibit MSI at four or more markers. In some cases the germline mutation has been identified (unpublished data) (34). Normal tissue was genotyped for D2S123 alleles and the stability of each allele assessed in matched tumour DNA. Thirty of the 41 tumours exhibited mutation at one or more D2S123 alleles and overall 37 of the 82 alleles (45%) had mutations. There was a significant bias in the frequency of mutations at the short alleles compared with long alleles (Fig. 4). Of 53 small wild-type alleles identified, 32 (60%) displayed instability in the tumour tissue compared with only 5 of 29 (17%) large alleles (21 = 12.4, P = 0.00043). Thus, constitutional allele sequence is a determinant of the propensity for instability at a given locus in the presence of defective MMR. This has important implications for the classification of clinical material with respect to MSI status.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We studied a paradigm CA repeat microsatellite locus, D2S123, and have shown that a cell line derived from non-tumour tissue displays a mutator phenotype and that the resultant accumulation of mutations is predicted by host genotype at each allele. D2S123 is used routinely in tumour MSI analysis and shows inter-allelic sequence variability for an interruption within the repeat tract. By genotyping and direct sequencing of individual alleles, we have demonstrated that defective MMR results in insertion and deletions of CA repeat units, which accounts for the variation in size of mutant alleles. In addition, alleles without a TA interspersion within the repeat tract are significantly more susceptible to instability as a consequence of defective MMR. Equal numbers of long and short alleles and their respectively assigned mutants were identified. Furthermore, insertion and deletion mutants derived from both progenitor alleles were detected. Such findings argue strongly against the trivial explanation that these observations are due to PCR bias in detecting shorter mutants at this locus. The analysis of a cohort of MSI+ colorectal cancer patients provides further confirmation that patient genotype directly influences manifestation of a mutator phenotype and the likelihood of MSI status being scored accurately.
Other studies that have addressed differences between loci in human populations and in colorectal cancers also show that perfect repeats are more susceptible to instability (8,9,11). However, this study is the first to demonstrate that variant interruptions can occur between alleles at the same marker loci and that these have a marked effect on individual allele stability. We show that the stabilizing effects of a variant repeat is apparent even in the presence of defective MMR, supporting a previous report (8). These results suggest that different alleles at the same marker loci can display genotypic variation, substantially affecting susceptibility to mutation.
Several studies have suggested that the mechanism of microsatellite mutation in MSI+ tumours shares similarity with the evolution of such sequences in the genome (11,20). D2S123 population allele frequencies from both our own data and that of the CEPH database highlights higher frequencies of short alleles. The CEPH database (http://www.cephb.fr/cephdb/ ) documents six short D2S123 alleles compared with two long ones (Table 2). This would be expected if short alleles with perfect repeats are more susceptible to mutation as our results from MMR-defective cells suggest. These mutations could then be fixed as new alleles. The absence of many length variants of the long alleles is also consistent with our sequence data showing that long alleles invariably contain the stabilizing interspersion.
It is generally accepted that replication slippage is the major mechanism causing new mutations in microsatellites (35). The results presented here are consistent with models in which a variant interruption causes stabilization by encouraging the perfect realignment of the two strands following their dissociation during such DNA polymerase slippage. However, the possibility that interruption of the repeat tract may alter some unusual structure associated with the repeats and subsequently reduce the rate of slippage cannot be wholly discounted (8).
The majority of mutations occurring at CA microsatellites have been shown to involve small length changes in which one or two repeats are altered (8,10,36). Mutations of repeat tracts in cancer genes of MSI+ colorectal tumours characteristically display small frameshift mutations (37,38). The mutations identified here at the D2S123 locus also involve the gain or loss of one or two repeat units in the majority of cases. In the cell line lbl-1261, short mutant alleles always contained perfect repeats, whereas long mutant alleles consistently possessed a TA interruption. The absence of any short mutants with a repeat interruption, or long mutants without an interruption or of mutants with duplicated TA interspersions argues against the occurrence of large sequence alterations in excess of a few repeats. This is consistent with hPMS2 being involved in the repair of small insertion/deletion loops (39).
Defects in MMR result in genome-wide accumulation of mutations at repetitive microsatellite sequences. We have used a cell line derived from a non-tumor lineage with a dominant negative MMR mutation to investigate inter-allelic MSI. This system has distinct advantages over using cancer cell lines. Cancer cell lines are subject to selection pressures for mutations that provide the neoplastic cell with a distinct growth advantage, thus evolving clonally and making them largely homogenous for mutations at any given microsatellite. In addition they accumulate multiple mutations and abnormalities, making the dissection of events resulting directly and exclusively from MMR defects difficult.
These data show that host genotype at CA repeat sequences can influence the ability of available marker sets to assign MSI status to any individual tumour. Therefore, these findings have important clinical relevance regarding MSI screening strategies and the effects of individual patient genotype on these analyses. Tumour MSI status is used to determine whether HNPCC may be discounted or whether analysis of MMR genes is required. Additionally there have been a number of reports indicating that MSI status may be used as a predictor of survival and can be employed as a clinical tool with which to give patient survival estimates (40–41). An accurate assessment of MSI status is therefore of critical importance. The other four microsatellite markers in the recommended panel (29) are entered in GenBank as uninterrupted (CA)n or (A)n repeats. However, the CEPH database (http://www.cephb.fr/cephdb/ ) indicates that they are polymorphic for different sized alleles. Such analysis by length may mask further underlying individual genotype differences at these markers. In any case, the phenomenon described here for D2S123 alone is of importance and the influence of patient genotype at this particular marker may be particularly critical in the diagnosis of borderline MSI cases. The data from this study highlight the need for a well-characterized set of diagnostic markers in which allelic variance and the intrinsic effect on stability is well understood. Suggestions that one or a few microsatellite markers are sufficient to assess MSI status should perhaps be treated with caution, especially when there may be significant implications for both the patient and the family (29,30,42). The marker studied here is logged in GenBank as an interspersed microsatellite. However we have shown that this does not reflect the underlying complexity of this locus. Many other markers may share similar intrinsic variances in allelic stability and the phenomenon may be widespread.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lymphoblast cell lines and tumour samples
Epstein–Barr virus (EBV) transformed lymphoblast cell lines were cultured from a healthy control individual (lbl-c5) and from the non-tumour tissue of a patient with colorectal cancer who had a family history of the disease (lbl-1261). Cell line lbl-1261 is derived from patient 6 referred by Parsons et al. (31) and displays a mutator phenotype due to a dominant negative mutation in hPMS2 (31,43).
Matched tumour and normal DNAs were previously isolated from 41 colorectal cancer patients shown to have defective MMR and a mutator phenotype. The causative MMR gene mutations have been defined in a number of cases (unpublished data) (34).
All patients with suitable material available whose tumours fulfilled MSI criteria were analysed in this study, regardless of whether or not D2S123 showed length variation in tumour DNA. There is potential bias because MSI criteria include D2S123 genotyping and so some patients with homozygous long alleles may be underscored with respect to MSI status. However, this does not impact adversely on the findings of these studies.
SP-PCR
We devised an assay to genotype D2S123 alleles, using an SP-PCR strategy in order to investigate the mutational behaviour of individual alleles. A similar approach has been used previously to detect mutations within populations of wild-type alleles (44,45). DNA from EBV transformed cell lines was diluted to a final concentration of 15–20 pg per PCR reaction to give up to three input molecules of each D2S123 allele per amplification (assuming 6 pg of DNA per diploid genome). Due to the dilute nature of the DNA, alleles were detected in 30% of analyses. Fluorescently labelled D2S123 primers were used in all PCR reactions (31). PCR amplifications were performed in a final volume of 25 µl. Final reaction concentrations were 1x PCR Buffer II (Boehringer, Mannheim, Germany), 0.2 mM dNTPs, 3.7 pM oligonucleotide primer and 0.87 U of Expand high fidelity PCR system enzyme mix. Reactions were prepared in 96 well plates. DNA-free controls were prepared in 16 of the wells in each plate and positive controls containing 100 ng of cell line DNA were prepared in 2 wells in every plate. Amplification was performed using an Omnigene PCR system thermal cycler (Hybaid) at 94°C for 3 min for 1 cycle, 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 35 cycles, 72°C for 5 min for 1 cycle. Two microlitres of each PCR reaction, including positive and negative controls, were analysed on an ABI 310 Automated Genetic Analyser, using Genescan software. For each DNA sample 100–250 SP-PCR products were generated and analysed. The frequency of mutant alleles in each cell line was expressed as the number of alleles which were mutant in length divided by the total number of alleles detected (normal and mutant). Accordingly, percentages are not exact contents of cells with alterations, but relative values of alleles. Differences between MSI frequency in the two cell lines was evaluated by 2 test and significance taken at 5%. Mutant allele origin was determined by assignment to the progenitor allele closest in size according to previous studies and our own experiments (2,8,32). Differences in mutation frequency of each progenitor allele was evaluated by 2 test. To account for the possibility that some mutants did not derive from the progenitor allele closest in size, a further more stringent assessment was performed by assuming that only mutants of <216 bp were derived from the smaller allele.
Sequencing D2S123 alleles
D2S123 alleles were amplified from undiluted cell line DNA using non-fluorescent primers and the aforementioned high fidelity PCR system and electrophoresed on a 3% Nusieve GTG agarose (Bioproducts, Rockland, ME) 1% ‘Hi Pure’ low EEO agarose (Biogene, Kimbolton, UK) gel in 1x TBE buffer at 30 V overnight and individual progenitor alleles gel purified (Qiagen, Crawley, UK). Sequencing was performed using PRISM Ready Big Dye Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase, FS (Taq-FS; Perkin Elmer/Applied Biosystems, Branchburg, NJ) and Applied Biosystems DNA sequencer model 373A or 377, according to the manufacturer’s instructions. D2S123 alleles from lbl-1261 and lbl-c5 were cloned into the TA cloning vectors (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions. Genotyping and cycle sequencing of transformants was performed as above.
Allele-specific bias in MSI tumours
One hundred nanograms of normal and tumour DNA templates were used in 50 µl PCR reactions as described. Analysis of individual allele shifts was made by comparison of Genescan profiles on the ABI analyser from normal and tumour DNA. Whenever there were doubts about the veracity of a mutation we did not include it. This applied to cases where the patient harboured two short alleles. On occasions it was questionable as to whether one or both alleles had mutated. Such cases were scored as shifting at just one of the alleles. Mutation frequency may therefore be marginally underestimated if both alleles had mutated and the presence of any wild-type allele in the tumour was due to contamination from surrounding normal mucosa. Observed differences in mutation frequency at each allele were evaluated by a 2 analysis.
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ACKNOWLEDGEMENTS |
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We are grateful to Bert Vogelstein for cell line lbl-1261. We acknowledge expert technical assistance from Sheila McBeath. This work was supported by the following grants: Cancer Research Campaign (SP2326/0201), Scottish Health Department (K/MRS/50/C2723), Melville Trust, Edinburgh University Cancer Research Endowment Fund and Urquhart Charitable Trust. A.L.B. was funded by an MRC PhD Student Fellowship, S.M.F. by an RSE Personal Research Fellowship and M.G.D. by an MRC Clinician Scientist Fellowship.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 131 467 8439; Fax: +44 131 343 2620; Email: malcolm.dunlop@hgu.mrc.ac.uk
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