Human Molecular Genetics, 2000, Vol. 9, No. 12 1881-1889
© 2000 Oxford University Press
Unequal exchange at the Charcot–Marie–Tooth disease type 1A recombination hot-spot is not elevated above the genome average rate
1Molecular Biology Program, Center for Computational and Experimental Genomics, University of Southern California, 835 West 37th Street, Los Angeles, CA 90089-1340, USA, 2Department of Pediatrics, Box 356320, University of Washington, Seattle, WA 98195, USA and 3Department of Mathematical and Computer Sciences, Colorado School of Mines, Golden, CO 80401-1887, USA
Received 24 April 2000; Revised and Accepted 2 June 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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An increasing number of human diseases and syndromes are being found to result from microduplications or microdeletions arising from meiotic recombination between homologous repeats on the same chromosome. The first microduplication syndrome delineated, Charcot–Marie–Tooth disease type 1A (CMT1A), results from unequal crossing over between two >98% identical 24 kb repeats (CMT1A-REPs) on chromosome 17. In addition to its medical significance, the CMT1A region has features that make it a unique resource for detailed analysis of human unequal recombination. Previous studies of CMT1A patients showed that the majority of unequal crossovers occurred within a small region (<1 kb) of the REPs suggesting the presence of a recombination hot-spot. We directly measured the frequency of unequal recombination in the hot-spot region using sperm from four normal individuals. Surprisingly, unequal recombination between the REPs occurs at a rate no greater than the average rate for the male genome (~1 cM/Mb) and is the same as that expected for equally aligned REPs. This conclusion extends to humans the findings in yeast that recombination between repeated sequences far apart on the same chromosome may occur at similar frequencies to allelic recombination. Finally, the CMT1A hot-spot stands in sharp contrast to the human MS32 minisatellite-associated hot-spot that exhibits highly enhanced recombination initiation in addition to positional specificity. One possibility is that the CMT1A hot-spot may consist of a region with genome average recombination potential embedded within a recombination cold-spot.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Charcot–Marie–Tooth disease type 1A (CMT1A) is a common inherited peripheral neuropathy that results from duplication of a 1.5 Mb segment on the proximal short arm of chromosome 17 (1–7). The duplicated region contains a dosage-sensitive myelin gene, peripheral myelin protein-22 (PMP22). The duplication arises by meiotic unequal crossing over between two almost identical (>98%) 24 kb repeats (CMT1A-REPs) located on either side of the 1.5 Mb region. The unequal recombination event occurs between a flanking proximal CMT1A-REP on one homolog misaligned with a distal CMT1A-REP on the other homolog. The reciprocal deletion product of this recombination event is responsible for the mild and less frequently diagnosed genetic disease, hereditary neuropathy with liability to pressure palsies (HNPP) (8,9).
Studies using DNA from CMT1A and HNPP patients showed that unequal crossing over is not uniform throughout the 24 kb CMT1A-REPs (8,10–16). Most events fall in a region <1 kb in length that has been defined as a recombination ‘hot-spot’. There is also a marked sex bias in the origin of the CMT1A duplication found in sporadic cases. De novo CMT1A duplication mutations almost always arise from an unequal recombination event in male gametogenesis (17–19).
Although positional specificity in CMT1A unequal recombination is well established, no direct genetic measurements of the unequal recombination frequency leading to CMT1A duplication chromosomes are available. Indirect frequency estimates could be based on population data on disease prevalence but would be subject to varying degrees of ascertainment bias. Using sperm samples from four unaffected donors, we directly determined the unequal recombination frequency between the CMT1A-REPs using a highly specific PCR assay that was confirmed by DNA sequencing. Surprisingly, we found that unequal recombination between a proximal and distal REP occurs at the male genome average rate (1 cM/Mb) and is not elevated above that expected for allelic recombination between two proximal (or two distal) REPs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Direct estimates of the CMT1A unequal recombination frequency in normal individuals
A PCR assay (Fig. 1) was used to detect unequal recombination within an ~1.4 kb region that includes the recombination hot-spot of the 24 kb REPs. The assay was designed to detect a single unequal recombination junction fragment in a sample of total sperm DNA from unaffected donors. Single molecule detection was achieved using two rounds of hemi-nested PCR. In each round, one proximal REP-specific and one distal REP-specific primer were employed. The design of these primers was based on sequence data of a cloned proximal and a cloned distal REP that revealed sites where a particular nucleotide(s) appeared specific to one or the other REPs (12) [cis-morphisms (20)]. We refer to these sites as signature sites. The distal REP-specific primers made use of a 1 or 3 nucleotide sequence difference and the proximal REP-specific primers a 4 base insertion/deletion difference between the proximal and distal REPs. The expected size of the PCR product is ~1.4 kb. The orientation of the primers allows detection of the junction fragment associated with the duplication, but not the deletion crossover product.
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The frequency of rare genetic events can be estimated using a PCR-based ‘counting’ assay (21,22). Table 1 summarizes our analysis of 842 aliquots of sperm DNA from the four unaffected donors. For each donor, the fraction of PCR reactions that gave the 1.4 kb PCR product was used to estimate the average number of chromosome 17 templates that contain a junction fragment. Each PCR reaction used 200 ng of sperm DNA (~66 000 copies of chromosome 17). At this dilution, the number of unequal recombination junction fragments is low enough that it varies from tube to tube according to a Poisson distribution with mean 66 000
, where is the unequal recombination frequency. It follows that the probability that a tube will contain no unequal recombination junction fragments is e–66 000. The unequal recombination frequency is estimated by setting this probability equal to the observed proportion of tubes that did not give the 1.4 kb product. If the true frequencies are assumed to be the same for all subjects, the estimate of the common frequency is 1.71 x 10–5, with a 95% confidence interval (CI) 1.56–1.85 x 10–5. In fact, the estimated mutation frequency of subject H (2.59 x 10–5) is different from the other three (P = 9.15 x 10–7). Under the assumption that A, C and L share a common frequency, the estimate of the overall frequency is 1.50 x 10–5, with a 95% CI 0.35–1.86 x 10–5.
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Figure 2 shows typical PCR results on sixty 200 ng sperm DNA samples from one of the four donors (A). Sixty-three percent (38/60) of the aliquots gave the 1.4 kb PCR product expected from amplification of a CMT1A unequal recombination junction fragment. Control experiments demonstrated the lack of potential artifacts such as ‘jumping PCR’ or PCR mispriming involving the unrecombined proximal and distal REPs that were present at much higher concentrations in each aliquot. PCR was carried out using clones of documented unrecombined REPs. No product was seen in samples containing equal amounts of cloned proximal and distal CMT1A-REPs equivalent to the number found in ~66 000 copies of chromosome 17. If DNA from a CMT1A patient diluted to contain about three copies of the unequal crossover product was added to these controls the 1.4 kb product was observed. Finally, aliquots of 200 ng of somatic DNA from four colon tumor cell lines also gave no product (data not shown). These controls confirm that observation of a 1.4 kb PCR product indicates the presence in the aliquot of a junction fragment that arose by a de novo unequal recombination event.
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At the time when our unequal recombination-specific primers were designed, sequence data on only a single unrecombined proximal and distal REP were available (12). Since then, studies on additional individuals (11,14) (see Materials and Methods) have shown that the particular nucleotides found at signature sites are not always unique to either the proximal or distal REPs in the population. Due to this individual variation it is critical (see Materials and Methods) that control experiments designed to test for mispriming and jumping PCR artifacts use cloned unrecombined REP regions from the same individual used for estimating the unequal recombination frequency.
We obtained clones of the hot-spot region from the unrecombined REPs of each of the four donors. Every donor had two different alleles at both the proximal and distal REP hot-spots. Equal amounts of each donor’s two different unrecombined proximal alleles and two different unrecombined distal alleles were mixed to yield the equivalent of 66 000 chromosome 17s. In each case no amplification of the 1.4 kb junction-specific PCR product was observed unless DNA from an individual with a CMT1A duplication chromosome was also added (data not shown.
Confirmation of the specificity of the PCR assay also came from cloning and DNA sequence analysis of a sample of the unequal recombination products chosen from three of the four donors (also see below). In every case the sequence data confirmed that the template for the PCR product was a chromosome that experienced an unequal recombination event in the hot-spot region between one unrecombined proximal and one unrecombined distal REP.
Location of the unequal recombination events in normal individuals
To locate the positions of unequal recombination within the hot-spot we compared the DNA sequences of each donor’s unrecombined REP alleles to the sequence found in the individual junction fragments from that donor. The junction fragment PCR products were cloned and multiple clones from each product were individually sequenced. A total of 55 of the 842 PCR products (~14%) were examined.
The 1.4 kb hot-spot region contained 12 potential signature sites that could be used to locate the position of the unequal recombination events. The remainder of the hot-spot region is identical in sequence. Excluding these 12 signature sites, Taq polymerase misincorporation or DNA sequencing errors were found at a frequency of ~0.27% among multiple clones that were sequenced. If applied to the 12 variable sites, this low mistyping frequency would not contribute to any significant errors in locating the recombination junctions.
Figure 3 shows the distribution of unequal recombination events across the 1.4 kb region for three sperm donors. The events fall into several different categories. The most common events (46/55) show a unique transition from proximal to distal REP sequences. The resolution of the location of this transition point in each individual is a function of which of the 12 signature sites are actually informative. This depends on the particular alleles at the two proximal and two distal REPs and on which REPs are involved in the unequal recombination event since there are two possible unequal pairing configurations. The remaining nine junction fragments are characterized by additional complexity. Seven were accompanied by gene conversion-like events similar to those suggested in studies of CMT1A and HNPP patients (11,23). Interestingly, five of the seven conversion-like events that we detected (71%) were associated with unequal recombination breakpoints in the nucleotide 3611–3780 region whereas this interval accounted for only 24% of all the events. Finally, four unequal recombinants (donors A and L) showed a nucleotide substitution at signature site number 5 (nucleotide position 3158). Although all four unrecombined proximal and distal REPs in both of these individuals contained the T allele at this site, only the other nucleotide known to exist at this position (A) was found in these four unequal recombinants. A de novo mutation associated with the unequal recombination event perhaps using a related sequence as a template could explain these events.
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A point mutation at signature site 5 is shown as a black box. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The frequency of unequal recombination between the CMT1A-REPs
CMT1A is one of the most fully characterized human diseases that originate from recombination between large homologous repeats on the same chromosome [citations in Potocki et al. (24)]. To study the details of meiotic unequal recombination between the CMT1A-REPs, a PCR assay was used to detect novel junction fragments in total sperm DNA. Sequence analysis confirmed that each PCR product was the result of an unequal recombination event between the sperm donor’s unrecombined proximal and distal REPs. Control experiments showed that the 1.4 kb products were unlikely to be artifacts of ‘jumping PCR’ or mispriming on unrecombined templates.
The PCR assay was designed to detect a junction fragment associated with unequal crossing over and leading to the CMT1A duplication. The primer orientations do not allow the reciprocal junction fragment associated with the deletion chromosome to be observed. However, the assay is not limited to detecting duplication chromosomes. Some unequal recombination events unassociated with crossing over but resolved as gene conversions may also be detected if a resolution event takes place in the 1.4 kb region. Therefore, the frequency estimate for events that lead specifically to the CMT1A duplication chromosome in unaffected individuals can be no greater than the observed unequal recombination frequency measured in our assay (~1.50 x 10–5). However, we argue below that most of the events that we detected were the result of unequal recombination leading to duplication.
The prevalence of CMT disease is thought to be 1 in 2500 (25). The fraction of CMT patients that are diagnosed with CMT1 is ~0.7 (5,26). Between 70 and 90% of CMT1 patients fall into the duplication-carrying CMT1A category. Among CMT1A patients it is estimated that 5–26% are sporadic (5,17,27,28, and references therein). The product of these probabilities is an estimate of the CMT1A duplication frequency due to unequal recombination throughout the whole 24 kb REP. These values range from 0.98–6.5 x 10–5 although considerable uncertainty remains due to ascertainment bias.
The above frequency estimate can be compared with the PCR based assay once the latter is corrected for the fact that only 75% of the CMT1A crossover junctions in patients occur within the hot-spot region we examined (10–16). This correction to the PCR data gives a maximum duplication crossover frequency of ~2.0 x 10–5 over the whole 24 kb REP.
The similarity between the patient-based and PCR-based frequencies suggests that most of the unequal recombination events detected by PCR involved an unequal crossover and formation of a duplication chromosome. The PCR assay is capable of detecting unequal recombination events unaccompanied by crossovers (gene conversion) as well as unequal crossovers leading to duplications. If gene conversion events were much more common than crossovers, the total frequency of events detected by PCR should have been much higher than the patient-based estimate that only counts de novo CMT1A duplications.
We did not examine sperm DNA from fathers of de novo CMT1A patients. It is possible that these unaffected fathers have much higher frequencies of unequal recombination than our four sperm donors. However, any such difference must be small since the patient-based frequency estimate is not far greater than that determined by PCR studies on our four donors. The similarity between the two estimates are also consistent with the idea that there are few negative consequences of the increased dosage in genes within the 1.5 Mb duplication region during development.
Does the 1.4 kb region of the CMT1A-REP undergo recombination more frequently than any average 1.4 kb piece of human DNA?
If a human chromosome segment of known length has a recombination fraction greater than expected based on the genome-average relationship between physical and genetic distance, the interval is defined as a recombination hot-spot. For males, the genome average relationship is 0.96 cM/Mb (29). The human pseudo-autosomal region (PAR1) is a well-known example of a hot-spot (30–32). This ~2.6 Mb segment appears to undergo crossing over during every male meiosis, whereas it would be expected to be recombined in only 2.6% of the gametes. Recent studies on PAR1 have compared a detailed radiation hybrid map of a series of PAR1 genetic markers with a high resolution genetic map of these same markers generated by single sperm typing (33). Data on eight intervals averaging ~380 kb in size have recombination fractions per unit of physical distance 13- to 70-fold greater than the genome average.
Another example comes from recent studies on the human MS32 minisatellite locus. A recombination hot-spot, ~1.5 kb in length and primarily located outside of the minisatellite sequence itself, contained subregions on the order of 200–300 bp with up to an ~110-fold increase in recombination compared with the genome average rate expected for intervals that size (34).
Using the same definition, the 1.4 kb CMT1A hot-spot region does not exhibit enhanced initiation of recombination. At the genome average rate (~1 cM/Mb), the recombination fraction between two equally aligned 1.4 kb DNA segments is expected to be ~1.4 x 10–5. The observed unequal recombination fraction in the 1.4 kb hot-spot is ~1.5 x 10–5 [after correcting the observed unequal recombination frequency data in Table 1 for the fact that there are two different unequal pairing configurations (proximal 1–distal 2; proximal 2–distal 1) and that the PCR assay detects only half of the crossover products]. Thus, although the REPs are 1.5 Mb apart and differ by 1–2% in sequence, the observed unequal meiotic recombination fraction between the proximal and distal 1.4 kb hot-spot regions is the same as that expected for meiotic recombination between alleles of any average 1.4 kb DNA sequence in the human genome.
Only one other direct measurement of unequal meiotic recombination between duplicated sequences in humans is available to compare with unequal recombination at the CMT1A-REPs. Using a semi-quantitative PCR method (35), unequal crossing over within a 100 bp region leading to a 30 kb deletion between the steroid 21-hydroxylase gene and its pseudogene was estimated at a frequency of 10–5 to 10–6. We calculate that the expected equal recombination fraction within a 100 bp interval should also equal ~10–6. Experiments in transgenic mice designed to measure meiotic intra- and inter-chromosomal gene conversion events (36–39) show rates that vary from 10–3 to 10–6 but differ significantly from one another in the experimental setting.
Equal versus unequal recombination in yeast
That the frequency of equal and unequal meiotic recombination between DNA sequences might be the same is especially surprising in the case of the CMT1A region since the proximal and distal REPs are separated by 1.5 Mb. However, results in yeast show that unequal and equal recombination between dispersed sequences can often be equally frequent (40–43). In the most relevant published yeast studies, diploid strains carrying leu2 alleles at the LEU2 locus and at the MAT locus 110 kb away on the same chromosome were analyzed (44). The frequency of recombination between leu2 alleles at LEU2 was comparable to that between either leu2 allele at MAT and one at LEU2. Unequal crossover products between homologs were found as frequently as intrachromatid crossovers leading to deletions (45). It is interesting to note that compared with total chromosome length (human chromosome 17, ~92 Mb; yeast chromosome III, ~320 kb) the relative distance between the repeats involved in recombination is significantly greater for yeast (human 1.6% and yeast 34% of chromosome length).
Equal recombination between the CMT1A-REPs
The conclusion that the 1.4 kb region is not a recombination hot-spot rests on a comparison between a measured unequal recombination frequency and an expected value based on the genome-wide average rate for equal recombination. Experimental data on the recombination fraction between equally aligned REP alleles in the 1.4 kb hot-spot interval are not available for comparison with our results. Recombination over such a small interval cannot be obtained using pedigree analysis although in certain individuals the nucleotide sequence differences between two proximal (or two distal) REPs may eventually permit a PCR based assay similar to the one described here.
Estimates of recombination within the 1.5 Mb interval between the REPs do exist. In one study, two probes within 1 Mb of each other were mapped. The male recombination fraction (most relevant here since de novo CMT1A duplications originate almost exclusively in male meiosis) was 4% (46). This is four times greater than the genome average rate and was taken to suggest that the region was prone to meiotic recombination, perhaps due to the CMT1A-REPs (4). Although standard human linkage maps can give highly accurate data on gene order, their power to estimate recombination fractions at high resolution is limited. In fact the recombination estimate of 4% was based on a maximum of 102 meioses for which we estimate a 95% CI of 1.6–10.2%.
We cannot formally exclude the possibility that, when equally aligned, the 1.4 kb hot-spot region has a greater recombination fraction than expected from the genome average. If this were true, the lower observed frequency of unequal recombination could be explained by the large physical distance between the REPs and their nucleotide sequence divergence. Under this hypothesis it would only be a coincidence that the observed unequal recombination frequency equals that expected from the genome average rate. A more detailed understanding of equal recombination in both males and females across the CMT1A region will be available when high resolution linkage data can be analyzed in the context of the definitive DNA sequence of this chromosome region.
Positional preference for recombination within the CMT1A-REPs
Despite the absence of evidence supporting enhanced initiation of unequal recombination in the 1.4 kb CMT1A-REP region, there is little question that the position of the unequal crossovers within the ~24 kb CMT1A-REP is highly restricted to this small sequence. Initially, 77% of the CMT1A and HNPP unequal crossovers in CMT1A patients were mapped within a 7.9 kb interval (10). Subsequent studies narrowed down the region to an internal 3.2 kb and then to a 1.7 kb fragment (12,15). More recently, the hot-spot was localized (according to GenBank accession no. HSU41166) to a 557 bp interval [from bp 2886 to 3442 (11)]; to a 741 bp stretch from bp 2603 to 3343 (14) and a 670 bp region from bp 2412 to 3081 (13). Almost all of the 55 de novo unequal recombinants that we analyzed occurred between 2603 and 3780 bp, similar to these earlier studies. We note that due to the nucleotide variation at the REP signature sites, knowledge of each individual’s unrecombined REP sequences is preferred over the use of REP consensus sequences to obtain the most accurate localization of recombination junctions.
One plausible explanation for positional specificity of unequal recombination within the CMT1A-REPs argues that the degree of uninterrupted sequence identity between the proximal and distal REPs in different regions determines the likely position of the crossover (11,14). This explanation is based on the well established relationship between mammalian recombination efficiency and number of base pairs of interrupted sequence identity (47–49). This idea could be further tested by comparison of larger numbers of unequal crossover products from CMT1A patients or unaffected sperm donors with the unrecombined REP sequences of the parents or donor, respectively. Sperm typing has the advantage that many recombination products could be obtained from individual donors specifically chosen with respect to the desired lengths of uninterrupted sequence identity.
The minisatellite, Chi and mariner-like transposable element (MITE) sequences found in or close to the 1.4 kb CMT1A-REP region may be involved in positioning recombination events within the CMT1A-REP (7,11,12,14,50,51). For example, the ~24 kb CMT1A-REP may take on a chromatin conformation unfavorable to recombination. The close proximity of the minisatellite, Chi or MITE sequences might enable the 1.4 kb region to exist in a chromatin conformation that allows recombination to occur. Under this hypothesis the 1.4 kb region could be a sequence island with a genome-average recombination potential surrounded by regions with a significantly lower recombination potential than the genome average (a recombination ‘cold-spot’).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Sperm DNA.
Sperm samples (A, C, H, L) from four healthy anonymous Caucasian donors (age 22–28 years) were kindly provided by California Fairfax Cryobank (Los Angeles, CA). DNA was extracted from washed sperm by sodium dodecyl sulphate (SDS), proteinase K and dithiothreitol incubation, phenol extraction and ethanol precipitation (52).
PCR assay
Previous CMT1A duplication-specific assays were developed to amplify the duplication junction fragment in affected individuals (11,13,14). We modified this assay to detect single de novo CMT1A unequal recombination product(s) in a pool of sperm DNA from unaffected donors. Trial experiments showed that using 200 ng aliquots, the number of unequal recombination junction fragments is low enough that it varies from aliquot to aliquot according to a Poisson distribution. Two rounds of nested PCR were used to enhance specificity and sensitivity. Additional specificity resulted from high annealing temperatures, matching the primer pairs with respect to annealing temperature and decreasing the annealing and elongation times. For the first round of PCR the initial denaturation was at 95°C for 3 min. The conditions for 30 cycles of PCR were: 30 s at 94°C, 10 s at 62°C and 70 s at 72°C. The final extension time was 10 min. In the second PCR round the conditions were the same except that the 72°C extension step was for 40 s and a total of 32 cycles were carried out. A GeneAmp 9600 thermal cycler was used for all reactions (PE Biosystems, Foster City, CA). PCR products were analyzed on 1.4% agarose gels.
First round PCR was carried out in 50 µl containing 10% volume of Mg2+-free 10x PCR buffer (500 mM KCl, 100 mM Tris–HCl pH 8.7, 1 mg/ml gelatin), 3.0 mM MgCl2, 0.4 µM each of primers D1 and P2, 0.2 mM of each dNTP,1.5 U of Taq DNA polymerase (Promega, Madison, WI), 0.15 µl of TaqStart antibody (Clontech, Palo Alto, CA) (Taq polymerase and TaqStart Antibody were mixed according to the manufacturer’s instructions) and 200 ng of sperm DNA. The reagents for second round PCR were the same as the first round except that primers D3 and P3 were at 0.2 µM and the Taq and TaqStart antibody were at 1 U and 0.1 µl, respectively. One microliter of first round product was used as template in the second round. The positions and sequences of the unequal recombination-specific PCR primers P2, P3, D1 and D3 are shown in Figure 1.
Amplification of unrecombined proximal or distal CMT1A-REP sequences made use of primers P1 and P2 or D1 and D2, respectively. Ten nanograms of genomic DNA was amplified from each donor in a single round of 45 cycles using the same conditions described for the first round of PCR to detect the junction-specific fragment.
For each donor, control experiments were carried out using cloned unrecombined REPs from the same individual. This precaution turned out to be important. In a screen of sperm samples from 21 different donors, eight gave anomalous results and consequently DNA from these eight donors was not used to estimate the unequal recombination frequency. Further characterization of the eight anomalous samples suggested that every cell in these eight individuals contained a template that can be amplified using the junction fragment-specific primer set. It is unreasonable to conclude that 8/21 (38%) unaffected sperm donors carry a CMT1A duplication chromosome. A more likely explanation is that these eight individuals carry one unrecombined REP allele with proximal-specific and distal-specific nucleotides at our primer binding sites (see below) that fortuitously mimics the junction fragments that can be detected with the unequal recombination-specific primer set.
Identifying recombination hot-spots
Using 20 ng of sperm DNA, the unrecombined proximal CMT1A-REPs of subjects A, C, H and L were amplified using proximal-REP-specific primers P1 (5'-GGATTCAAAGATATTAGTGTTAT-3') and P2 and the unrecombined distal REPs using distal-REP-specific primers D1 and D2 (5'-AAGTTAAAGGGGTAACTAGAGA-3'). The 2.3 kb PCR products were cloned (TOPO TA Cloning kit; Invitrogen, Carlsbad, CA) and sequenced in the region covering the 1.4 kb hot-spot. Under our PCR conditions amplification was proximal REP- or distal REP-specific as judged by experiments showing that a documented unrecombined proximal REP clone only gave amplification with the proximal-specific PCR primer pair. The PCR products obtained using the distal-specific primers but not the proximal-specific primers could be digested with EcoRI which recognizes a site specific to only the distal REP hot-spot region. The specificity of the unrecombined REP PCR was also confirmed by DNA sequencing. Five or six distal and five to seven proximal clones were sequenced from each individual. Three one-pass sequencing runs (PE 377) were carried out on each clone and analyzed using GCG and/or programs developed at the Baylor Human Genome Sequencing Center (http://www.hgsc.bcm.tmc.edu/tools/ ).
Five different unrecombined distal REP alleles were found in the four sperm donors. Four of the five contained from one to three adjacent proximal REP-specific nucleotides among the 15 signature sites that we sequenced (data not shown). Three different proximal REP alleles were found. Each contained one to three adjacent distal REP-specific nucleotides among the 15 proximal-REP signature sites. These data suggest that unequal recombination not associated with crossing over (gene conversion) has occurred between proximal and distal REPs. Alternatively, reversion of a CMT1A duplication by an intra-chromatid recombination or recombination between a normal REP and a CMT1A chimeric REP could explain the existence of these different allele types in the population.
The unequal crossover PCR products were cloned and at least two clones sequenced. If they were identical at the signature sites then no additional clones were examined. If there was a difference at these sites between the two clones then additional clones were sequenced to confirm the existence of more than one unequal recombination product in the sample. It is not surprising that a sample might contain two unequal crossover products given that there were on average 1.6 such products per sample.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the NIH (N.A., R37GM36745; P.F.C., R01NS38181), the Muscular Dystrophy Association (P.F.C.), the Swiss National Science Foundation (M.P.K.) and NSF grant DMS 98-72005 (W.N.). The authors wish to acknowledge Dr Jim Lupski at the Baylor College of Medicine and Dr Larry Reiter at the University of California at San Diego for their helpful comments on the manuscript.
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FOOTNOTES |
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+ Present address: University of Basel, Department of Research, ZLF 406, Hebelstrasse 20, 4031 Basel, Switzerland §To whom correspondence should be addressed. Tel: +1 213 740 7675; Fax: +1 213 740 8631; Email: arnheim@usc.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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