Human Molecular Genetics, 2000, Vol. 9, No. 8 1239-1244
© 2000 Oxford University Press
Crossover breakpoint mapping identifies a subtelomeric hotspot for male meiotic recombination
Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK and 1Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK
Received 7 February 2000; Revised and Accepted 7 March 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Segregation analysis of CEPH and other pedigrees yielded six paternal crossover breakpoints in the ~85 kb interval between the minisatellite loci D16S309 (MS205) and D16S83 (EKMDA2) in 16p13.3. Three crossovers were mapped to within the same small (<3 kb) interval, which does not co-localize with any tandem repeat array or expressed sequence. Haplotyping of loci harbouring single nucleotide polymorphism (SNP) markers in this interval confirmed the exchange of flanking markers in the three recombinant individuals. Sequence analysis revealed the presence of recombination-associated motifs and binding sites for the protein translin. Haplotyping of 108 individuals from three European populations at four loci harbouring SNPs showed substantial linkage equilibrium across this interval. Hence molecular and population genetic data are consistent with the presence of an intense male-specific recombination hotspot at this locus.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Variation in the meiotic recombination rate throughout the human genome is a well documented but poorly understood feature of human genetics. Regions with elevated rates, suggested by linkage disequilibrium studies and meiotic crossover breakpoints, at the dystrophin (1), ß globin (2), insulin (3) and the PGM1 (4) loci, and within the MHC (5), have been extensively analysed to try to identify common features of such ‘hotspots’. Subtelomeric regions of dicentric autosomes show a consistent male-specific enhancement in recombination rate and are rich in highly variable minisatellites. The combination of these properties recommend subtelomeric regions for the study of the features required for hotspot activity. In this study we present data on the localization of a subtelomeric crossover hotspot which shows an enhancement of at least 300-fold above the genome average. Sequence analysis of the breakpoint hotspot and its surroundings is used to address the question of sequence motifs and properties that may explain the activity of this region. Population genetic studies reveal levels of linkage equilibrium consistent with a highly active recombination hotspot.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Segregation analysis
The polymorphic minisatellite loci D16S309 (MS205) and D16S83 (EKMDA2) map ~85 kb apart in 16p13.3, ~1.3 Mb from the telomere (Fig. 1a). Segregation data in all 40 CEPH families were analysed using CRIMAP (6) at the Human Genome Mapping Project (HGMP; Hinxton, UK) to identify recombinant progeny. In rare cases where either MS205 or EKMDA2 and their adjacent minisatellites were uninformative, segregation data from more distal markers was used to infer the absence of a crossover in this interval. In addition to the CEPH families, 13 families from a mapping study and two families from the ECACC repository were typed. In total six paternal recombinants (Fig. 1b) were verified in 348 informative paternal transmissions. No recombinants were seen in 384 informative maternal transmissions. This suggested a paternal crossover rate in the 85 kb interval between MS205 and EKMDA2 of 1.7% (0.34–3.1%, 95% confidence interval), ~22-fold elevated above the paternal genome-wide average of 0.9 cM/Mb (7).
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Fine-scale mapping
Fine mapping with additional DNA polymorphisms was used to narrow the localization of the six known recombinants (Fig. 1b). The breakpoints of three of the six known recombinants mapped to a maximum common interval of <3 kb (inset, Fig. 1b). The other three recombination breakpoints were localized to distinct intervals. Recombinants 135009 and LF10.1 were localized to intervals of ~9 and 6 kb, respectively. Recombinant 136210 can only be localized to an ~57 kb interval, due to the lack of informative markers in the father. Thus, half of the known recombination events in the MS205–EKMDA2 interval are clustered within 3 kb. Monte-Carlo simulations indicate that such clustering can occur by chance in ~6% of cases, even given a completely uniform rate of crossover across the 85 kb interval. However, given that our mapping with flanking heterozygous markers gives the maximum interval within which the crossovers must occur, this is a conservative estimate of the significance of the observed clustering. Sequencing telomeric to the critical interval yielded 8.4 kb of contiguous sequence from the fathers of all six recombinant progeny, and the characterization of 22 novel single nucleotide polymorphisms (SNPs). In the case of the three clustered recombinants the sequence of the critical region was determined to ensure consistency with the segregation data. In all three cases the recombinant interval contained no sequence variation, apart from the previously characterized SNPs.
To confirm the phase of the segregating haplotypes at the four critical SNPs, and thus the implied breakpoint localization, the sequence data were used to design allele-specific polymerase chain reaction (PCR) primers for each SNP. These primers were designed to specifically amplify only one allele of each biallelic polymorphism under comparable conditions (control lanes 1–3, Fig. 2). The four pairwise combinations of each pair of SNPs were then used in double allele specific PCR (DAS-PCR). In each case the recombinant progeny (C) inherits an intact maternal (M) haplotype, and a novel recombinant haplotype (arrows, Fig. 2) derived by crossing over between the paternal (F) haplotypes.
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Sequence analysis
The segregation data for this region of the MS205–EKMDA2 interval strongly suggest the presence of a male crossover breakpoint hotspot. As a first step in the comparative sequence analysis of this breakpoint hotspot, the 8.4 kb of sequence determined in the mapping stage was placed in a wider genomic context, to precisely locate it within the MS205–EKMDA2 interval. Extensive finished genomic sequence is available for this region, enabling the construction of a framework sequence map (Fig. 1c). Two known genes have been assigned to this interval (Fig. 1c). At the telomeric end of the contig is a region showing identity (98.5%) to the somatostatin V coding sequence (8). The first nine exons of the
1H subunit of a novel human voltage-dependent T-type calcium channel (9) map on to the genomic sequence centromeric to the CpG island in the centre of the MS205–EKMDA2 cosmid contig. Expressed sequence tag hits to the second exon confirm the expression of this sequence. Other (weakly) predicted exons (ex1–5, Fig. 1c) from the region were not amplified from cDNA libraries or by RT–PCR from fetal RNA, suggesting that there are no other expressed sequences in this region. Hence the crossover breakpoint hotspot is not closely associated with any identifiable coding sequences.
Sequence motifs
Further sequence analysis was restricted to the critical 3 kb region and the 6 kb centromeric and telomeric flanking sequences. This ~15 kb of sequence was searched for consensus matches to sequence motifs previously associated with recombination hotspots, and binding sites for proteins involved in recombination (Materials and Methods). This search revealed a single 
site and three translin binding sites (T1–3) (Fig. 1c). In sequences of this region’s GC composition (66.3%), sites and translin sites are expected to occur by chance approximately once in 28.5 and 1200 kb, respectively. Hence the occurrence of a single site is unremarkable. By contrast three translin sites, two of which overlap in inverted orientation, seem highly unlikely to occur by chance. In addition a 9 bp perfect inverted repeat (9 bp IR, Fig. 1c), with similarity to part of the translin consensus, also occurs.
The ß globin recombination breakpoint hotspot is associated with the local functional origin of replication. Whether this implies a link between recombination and replication is unclear but the primary sequence in this region shows a marked propensity to unwind, i.e. to exhibit low helical stability. Hence helical stability of the 15 kb interval was investigated using the sliding window analysis implemented by the program THERMODYN (10) over the 15 kb test interval (software available at http:/mcbio.med.buffalo.edu/CMB/kowalski/dk.html ). 
G, the free energy difference (in kcal/mol) between duplex and single stranded states was calculated for 100 bp windows at 5 bp intervals, assuming a temperature of 37°C and ionic strength of 10 mM. This analysis indicated a region of low helical stability in the Fnu4HI–‘BspHI’ interval typical of DNA unwinding elements (DUEs) at human and yeast replication origins (11,12). In this region G varies from a local maximum of 150.1 kcal/mol, to a local minimum of 114.6 kcal/mol, and back to a local maximum of 155.2 kcal/mol, within 225 bp.
Potential DNA curvature and flexibilty (13), which may influence chromatin structure and thus DNA accessibility was analysed using the Bend.It web server: (http://www.icgeb.trieste.it/dna/bend_it.html ). A region of moderate curvature and low flexibility, which may represent a stable curved structure occurs at both the ‘MboII’ and ‘BspHI’ polymorphisms.
Translin binding
As noted above, the FokI–Fnu4HI sub-interval contains two sequences that conform to the consensus binding sites of the single stranded (ss) DNA end binding protein, translin. These sequences overlap and are in inverted orientation. An initial approach to addressing the significance of this site was to establish whether it was bound in vitro by the translin protein. Translin is a sequence-specific ssDNA end-binding protein (14) that has been implicated in the binding of regions of ssDNA generated by resection of double strand breaks. Recombinant human translin (the kind gift of Dr Masataka Kasai) was used in binding reactions to radiolabelled oligonucleotides containing the putative binding site sequences (data not shown). One of the oligonucleotides, T2487, from the double site, shows strong sequence-specific binding. The other site, T2495, by contrast shows little, if any, sequence-specific binding. The third site, 4.7 kb centromeric to the ‘MboII’ polymorphism and outside the critical interval, was not tested.
Population studies
Given a male average genome-wide recombination rate of 0.9 cM/Mb (7), three recombinants in 348 informative transmissions suggest that the paternal recombination rate for this 3 kb interval represents an enhancement in excess of 300-fold. This estimate is based on a small number of events derived from pedigree information and consequently the error in this estimate may be large. However, such a large localized apparent increase in the rate of paternal recombination would be expected to have a profound effect on the association between polymorphic markers in this interval. The DAS-PCR assays developed to confirm the breakpoint localization were used to assess linkage disequilibrium between these polymorphisms. All four loci were haplotyped in a total of 108 individuals from three European populations. Pairwise disequilibrium measures (Fig. 3) were calculated for each of the three intervals. The D' statistic (15) was used, where 1 indicates complete association and 0 indicates linkage equilibrium. In the UK and Basque populations there is a pronounced depression in D' in the FokI–Fnu4HI interval, i.e. we cannot reject a null hypothesis of linkage equilibrium (16,17). In the German population this interval shows significant linkage disequilibrium, while it is in the Fnu4HI–‘BspHI’ interval that the null hypothesis cannot be rejected. Overall, this analysis indicates that in all three populations there are polymorphic loci in linkage equilibrium within the critical 3 kb interval, consistent with the presence of a region of intense recombinational activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fine mapping of crossover breakpoints in families requires not only large pedigrees, but also a high density of informative polymorphic markers, properties most often satisfied by regions containing genes of medical importance, as exemplified by the ß globin locus (2), the insulin locus (3), the DMD locus (1) and the TAP1/TAP2 locus in the MHC (5). In all these cases the effect of selection on haplotypic associations at these loci is unknown. In addition, the enhancements in recombination rates at these loci tend to be moderate (3- to 30-fold over the genome-wide average). A notable exception is the PGM1 locus (4) where clustering of breakpoints within a 12 kb interval suggests a paternal recombination rate of 66 cM/Mb, a 73-fold enhancement. However, the absence of identifiable coding sequences near the recombination hotspot reported here suggests that selection does not influence haplotypic associations in this region.
Unstable minisatellite loci owe their variability to polar inter- and intra-allelic recombination processes that in the case of MS32 (D1S8) extend into the flanking DNA to generate intense localized linkage equilibrium [i.e. constitute a minisatellite-associated recombination hotspot (18)]. Despite the proximity of two highly variable minisatellite sequences (MS205 and EKMDA2) in this region of 16p13.3, crossover breakpoints cluster within an interval of <3 kb, distant from either locus. This suggests that overall VNTR sequences are not major contributors to crossing over in this region.
Sequence analysis of the critical region and its flanking DNA revealed few motifs previously implicated in recombination activity. The occurrence of a single sequence is not surprising given the GC content of this region. The 15 kb test interval examined in detail contains three translin sites, whereas the expectation for a random sequence of this composition is 1 in 1200 kb. The telomeric flanking region from which we can exclude recombination events (Fig. 1a, from the telomeric end of the test interval to EKMDA2, ~15.5 kb) also contains two translin sites, suggesting that there may be significant enrichment for these sequences in the entire interval. However, the occurrence of two sites within the FokI–Fnu4HI sub-interval, in an inverted overlapping conformation, appeared sufficiently unlikely to warrant investigation. In vitro binding of recombinant human translin shows, however, that only one of these sequences is recognized by translin under the conditions tested. It should be noted that the site recognized (T2487) conforms to the majority consensus translin site, 5'-GCNC[A/T][G/C][G/C][A/T] N0–2 GCCC[A/T][G/C][G/C][A/T]-3', while the site that is not bound is of a minority non-repetitive form 5'-[C/A]TGCAG N0–4 GCCC[A/T][G/C][G/C][A/T]-3'. This strongly suggests that the sequence-specific binding propensity of translin is context-specific and that consensus sequences derived from chromosomal breakpoints may integrate other affinities not evident in vitro. Interestingly the sequence 5'-CTGCAG-3' occurs as part of a 9 bp perfect inverted repeat (5'-CTGCAGAGGCCTCTGCAG-3') within the Fnu4HI–‘BspHI’ interval, close to the region identified by helical stability analysis as a DUE.
DUEs are associated with and play a functional role at a number of replication origins in Saccharomyces cerevisiae (12). Their occurrence at the ß globin (19) and GABAA gene cluster (11) replication origins may suggest a similar role at some human replication origins. Alternatively, recombination initiation at replication origins may simply reflect DNA accessibility. In the case of the MS205–EKMDA2 hotspot it will be important to establish whether the identified DUE is associated with a functional replication origin, despite the absence of consensus origin sequences (20). The nascent strand abundance assay (21) provides a PCR based method for the detection of BrdU-labelled nascent strands, specific to the DUE region, in cultured cells.
The local changes in DNA curvature and flexibility predicted near the ‘MboII’ and ‘BspHI’ polymorphisms may be expected to disrupt chromatin structure. This could be assayed by observing patterns of nuclease sensitivity in chromatin. In vivo treatment of chromatin with specific (MspI) and non-specific (DNaseI) nucleases (22) will provide, after Southern blotting, a direct means of assessing hypersensitivity at this locus. However, whether the chromatin structure of this region in cultured cell lines reflects that observed in meiotic spermatocytes remains to be seen.
Finally, the hypothesis that the clustering of crossover breakpoints reflects localized recombination activity is supported by linkage equilibrium in this interval in three European populations. The fact that the three populations show different distributions of linkage disequilibrium within the critical (3 kb) region may result from the influence of population history on allelic associations. Present-day associations will reflect the sampling of ancestral lineages with various recombination histories, which may be sufficient to generate disparate allelic associations even in very recently diverged populations. However, it will be of interest to extend the haplotype analysis into the flanking regions to further characterize the extent of linkage equilibrium, particularly for the German population. Preliminary attempts were frustrated by the difficulty of maintaining discriminatory DAS-PCR over intervals of >3 kb in such high GC content regions. Furthermore, extended screening for substitutional polymorphism by sequence analysis of six unrelated individuals only yielded a further 18 variant sites in a total of 8.4 kb. This is in marked contrast to the extremely high nucleotide diversity near the TAP2 hotspot (23), and limits the resources available for further analysis of the region either by association or sperm DNA studies.
Concluding remarks
The hotspot of male-specific meiotic recombination reported here is distinctive in that it is not associated with coding sequences or tandem repeat arrays. Sequence analysis suggests that this region has the potential to form an open chromatin structure, which may facilitate the binding of DNA binding proteins involved in recombination, such as translin. Population genetic analyses confirm the impact of intense recombinational activity on allelic associations at this locus. This enigmatic region of elevated male-specific recombination may form a prototype for foci of crossover activity in subtelomeric regions of the human genome, where recombination rates are consistently enhanced in the male germline (7).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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SNP screening
High density (at least 10-fold coverage) small insert libraries were constructed from cosmids from the MS205–EKMDA2 interval (Fig. 1a) These arrayed libraries were probed with defined restriction fragments from particular cosmids enabling the isolation of small (~1 kb) sequence fragments from known locations that as PCR products were screened for SNPs. All the recombinants were paternal in origin and hence SNP screening was confined to the fathers of recombinant progeny. Genomic PCR products from these individuals were initially screened for heterozygous positions using 20 restriction endonucleases with 4 bp recognition sites. Enzymes with restriction patterns indicating the presence of a heterozygous SNP were then used in PCR–restriction fragment length polymorphism (RFLP) assays to determine the segregation. In the absence of RFLP assays direct cycle sequencing of the PCR products using 33P-labelled terminators (Amersham Pharmacia Biotech, Uppsala, Sweden) was used to screen for sequence variation. Sites that naturally destroyed/created recognition sites for commercially available restriction endonucleases were typed using PCR–RFLP assays. Sites that did not satisfy this requirement were assayed using modifying PCR primers adjacent to the heterozygous position that introduced convenient restriction endonuclease recognition sites at the end of the PCR product (indicated by the name of the restriction enzyme being placed in quotation marks). Segregation was then determined by PCR–RFLP assay.
Critical polymorphisms and DAS-PCR primers
‘MboII’. An A/C polymorphism assayed by amplification with flanking primers, one of which generates an MboII recognition site, followed by restriction. DAS-PCR primers: MBFA: 5'-TCTGTTTCCAAAACTCGCCTA-3'; MBFC: 5'-CTGTTTCCAAAACTCGCCTC-3'.
FokI. A naturally occurring RFLP resulting from a G/A polymorphism. DAS-PCR primers: FOFG: 5'-CCGTGTCCCCACCCCTCG-3'; FOFA: 5'-GCCGTGTCCCCACCCCTCA-3'.
Fnu4HI. A naturally occurring RFLP resulting from a G/A polymorphism. DAS-PCR primers: FNFA: 5'-TGCACAGGGAGCTCAGGCTA-3'; FNFG: 5'-GCACAGGGAGCTCAGGCTG-3'; FNRT: 5'-AGGGCAAAGGCACAGGGT-3'; FNRC: 5'-GGGCAAAGGCACAGGGC-3'.
‘BspHI’. An A/T polymorphism assayed by amplification with flanking primers, one of which generates a BspHI recognition site, followed by restriction. DAS-PCR primers: BSRA: 5'-GCCCGTCCCCGTGTGATGA-3'; BSRT: 5'-GCCCGTCCCCGTGTGATGT-3'.
Note, the third letter of each DAS-PCR primer name indicates the direction of extension (F, telomeric; R, centromeric). Appropriate assay combinations, for example MBFA/C FNRT/C or FNFA/G BSRA/T.
Sequence analysis
Finished sequence was combined with unfinished sequence downloaded from the Sanger Centre FTP site (ftp.sanger.ac.uk ) to construct a sequence framework. Sequence samples from known map locations on the cosmid contig (Fig. 1a) were used to verify the ordering of the constructed sequence contig, with particular reference to known minisatellites. The region containing the breakpoint hotspot is within the finished sequence of cosmid c381g6. Extending the contig centromeric of this cosmid requires the utilization of two large contigs from the cosmid c344f5, 00261 and 00929. The assembled contiguous sequence was analysed using the NIX analysis suite at the HGMP website (http://www.hgmp.mrc.ac.uk/ ). This application uses parallel analysis of genomic sequence with many established search techniques to aid gene identification. BLAST based searches of databases containing genomic, expressed and repeat sequences are combined with coding sequence prediction analyses (GRAIL, Fexon, Fgenes, etc.) providing a powerful consensus on coding potential.
Recombination/replication motifs
The test interval [the critical 3 kb interval and the flanking (5' and 3')] 6 kb was searched using the FINDPATTERNS utility of the GCG software suite, at HGMP. The motifs examined were: (5'-GCTGGTGG-3') from Escherichia coli (24), the ade6-M26 heptamer (5'-ATGACGT-3') from Schizosaccharomyces pombe (25), the long terminal repeat element (LTR-IS) motif (26) (5'-TGGAAATCCCC-3'), the retrotransposon LTR sequence (27) (5'-TCATACACCACGCAGGGGTAGAGG ACT-3') the XY32 homopurine–pyrimidine H-palindrome motif (28) (5'-AAGGGAGAARGGGTATAGGGRAAGAGGGAA-3'), the human minisatellite core sequence (5'-GGGCAGGARG-3'), two human hypervariable minisatellite sequences (29,30) (5'-GGAGGTGGGCAGGARG-3')5 and (5'-AGAGGTGGGCAGGTGG-3')5. The protein binding sites were that of pur (31) 5'-GGNNGAGGGAGARRRR-3' [which has a site near the ß globin hotspot (2)] and translin: consensus 1, 5'-GCNC[A/T][G/C][G/C][A/T] N(0–2) GCCC[A/T][G/C][G/C][A/T]-3', consensus 2, 5'-[C/A]TGCAG N(0–4) GCCC[A/T][G/C][G/C][A/T-3' [both associated with chromosomal breakpoints in T-acute lymphoblastic leukaemias (14)]. Sequences associated with replication origins were also examined: the human replication origin consensus 5'-WAWTTDDWWWDHWGWHMAWTT-3' (20) S.cerevisiae autonomously replicating sequence (ARS) consensus 5'-WTTTATRTTTW-3', S.pombe ARS consensus 5'-WRTTTATTTAW-3', consensus scaffold attachment regions, 5'-AATAAAYAAA-3', 5'-TTWTWTTWTT, WADAWAYAWW-3', 5'-TWWTDTTWWW-3' (32) and the topoisomerase II binding site (33) 5'-GTNWAYATTNATNNR-3'.
Statistical analysis of linkage disequilibrium
The probability of the observed haplotype distribution occurring by chance in the absence of linkage disequilibrium was assessed using a modification of Fisher’s exact test (16). The Markov chain implementation of this test, as featured in the Arlequin suite of programs (17) was applied to the haplotype data with a null hypothesis of linkage equilibrium.
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ACKNOWLEDGEMENTS |
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Thanks go to J. Attwood for help with linkage computing, P. Harris and N. Doggett for cosmids, N. Royle for sharing unpublished segregation data, D. Higgs and J. Flint for access to unpublished sequence data, R. Trembath for family DNA samples, E. Rogers for sharing disequilibrium data and Masataki Kasai for the gift of recombinant human translin, and advice on gel shift assays. Computing facilities were provided by HGMP. This research was funded by The Wellcome Trust (Grant no. 047113/Z/96/Z).
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FOOTNOTES |
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+ Present address: Department of Human Genetics, University of Michigan Medical School, Medical Science II M4708, Ann Arbor, MI 48109-0618, USA
§ To whom correspondence should be addressed. Tel: +44 115 9249924; Fax: +44 115 9709906; Email: john.armour@nottingham.ac.uk
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