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,1
,
* Scientific Institute IRCCS E. Medea, Bosisio Parini (LC), Italy;
Molecular Genetics Program, Center for Medical Sciences, Wadsworth Center, Albany, New York, USA;
Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena Foundation, Milan, Italy; and
Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York, USA
1Correspondence: IRCCS E. Medea, Via Don L. Monza 20, 23842 Bosisio Parini (LC), Italy. E-mail: msironi{at}bp.lnf.it
SPECIFIC AIMS
Deletions within the dystrophin gene (DMD) account for >70% of mutations leading to Duchenne and Becker muscular dystrophies (DMD and BMD, respectively). Deletion breakpoints have been reported to be scattered within large regions that also represent meiotic recombination hot spots, and no causative sequence or structure has been identified for induction of deletion events.
The aim of our study was to use a combined approach (physical analysis of yeast meiotic DNA, statistical analysis, and human molecular genetics techniques) to study the molecular events leading to deletion formation in the DMD gene major hot spot.
PRINCIPAL FINDINGS
1. A region in intron 47 induces DSBs during meiosis
The ability to induce DBSs during yeast meiosis was initially assessed for seven fragments from the DMD gene major hot spot. All human sequences were inserted within the HIS4 locus of Saccharomyces cerevisiae (Fig. 1
).
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We had previously identified a 1250 bp region in intron 47 (IVS47) where three deletion breakpoints were located (B1-B3 in the upper panel of figure Fig. 2
); three inserts (DMD47–1, DMD47–2, and DMD 47–3) were therefore designed to cover the regions surrounding the three breakpoints (Fig. 2)
. Three more inserts were derived from DMD intron 44 (IVS44), and each covered the region where a deletion breakpoint had previously been located. Finally, a control insert deriving from IVS 71 (where deletions are extremely rare) was cloned within HIS4. The rad50S mutation, which prevents processing of DSBs, was subsequently introduced into the yeast strains carrying HIS4-DMD alleles. Diploids were then obtained by mating with a rad50S strain.
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Physical analysis of meiotic DNA was performed through Southern blot and hybridization. Most meiotic recombination at the HIS4 locus occurs due to DSB formation at the HIS4 promoter (Site I). In all strains (see Fig. 1
), a PvuII enzymatic digestion and Southern hybridization with an XhoI-BglII fragment of the HIS4 coding region as a probe is expected to yield a band (2.4 kb) corresponding to the almost-complete wild-type (WT) HIS4 gene and a portion of BIK1 plus a fainter product corresponding to DSB formation at HIS4 promoter (site I, 
1.9 kb). Strains carrying heterozygous his4-DMD alleles are expected to yield at least two additional bands: one corresponding to the inserted allele and the other due to DSB formation at site I on chromosome containing the DMD insert (Fig. 1)
. Similar patterns are expected to derive from NsiI digestions (Fig. 1)
. A PvuII digestion of MSY14 (HIS4/his4-DMD47–3 rad50S/rad50S) meiotic DNA generated an extra band (1.5 kb) (Fig. 3
A). The size of this band suggests it originated through formation of DSBs within the DMD insert; moreover, the smeared appearance of the band suggests that breaks had occurred at multiple sites. No other strains showed any evidence of additional bands, and therefore of DSB formation (Fig. 3A-C
).
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2. A short region is able to generate DSBs
Since inserts DMD 47–3 and DMD 47–2 partially overlap (Fig. 2)
, we speculated that the sequence responsible for DSB formation resides in the nonoverlap region (186 bp). We constructed yeast strains carrying inserts DMD 47–4 and 47–5 (Fig. 2)
within the HIS4 locus as described above. In addition, the 186 bp nonoverlapping region was split into three smaller fragments (47–6, -7, and -8; Fig. 2
), and these were used to construct three additional strains. The 186 bp region induced DSBs, although with much lower frequency than the longer 47–3 fragment. Conversely, no DSB was observed when the 186 bp region was located at the 3' end of an insert or when it was split into three smaller fragments.
3. DSBs occur within the DMD-47 inserts
We wanted to determine whether DSBs occurred within the DMD inserts or in the flanking HIS4 sequences. The 0 h sample of DNY115 was digested with PvuII and SalI, then compared to the 24 h samples of MSY 14 and 35 digested with PvuII. Digested DNA was blotted and hybridized to either the right or left probe (see Fig. 1
). In case DSBs occurred on one side of the DMD insert, one of the two DSB-derived fragments from MSY 14 and MSY 35 would be equal to or smaller than the control fragments derived from DNY115 DNA; conversely, both fragments generated by the DSB were larger than the control DNY115 fragments, suggesting that DSBs occur within the DMD inserts.
4. Deletion breakpoints are clustered in intron 47
We retrieved from previous works all sequenced deletion breakpoints located in intron 47, so that 29 distinct locations were obtained. Analysis of breakpoint distribution was performed using kernel density estimates. Two IVS47 regions displayed breakpoint density estimates greater than the 95% envelope value deriving from sample simulations (Fig. 2)
: the region furthest 5' was centered around the DSB prone region; the second cluster was located downstream and contains four breakpoints. We consequently wanted to verify whether in an independent population of DMD/BMD patients carrying deletions involving intron 47, the frequency of breakpoints around the DSB prone region was higher than expected. We mapped breakpoint locations for 26 DMD/BMD patients with deletions involving intron 47. Three patients (11.5%) were found to have a deletion breakpoint mapping between markers IVS47C3 and IVS47A4; in particular, the three breaks were located in a region shorter than 2 kb centered around marker IVS47W3 (breakpoints B4-B6 in Fig. 2
). Given the size of intron 47 (54.22 kb), breakpoint occurrence in this region is 3-fold higher than would be expected if breaks were randomly distributed (3.7%). Deletion junctions were obtained for the three patients and are reported in Supplemental Fig. 1.
5. No evident association between DSB and secondary structure
The 47–3 region of IVS 47 was searched for motifs previously associated with recombination hot spots, and no recombinogenic motif was found to be over-represented within the DSB-prone region relative to either IVS47 flanking sequences (IVS47C3-A4 interval) or IVS71. The presence of direct, inverted, complementary, and mirror repeats was also assessed and no evident association was observed.
Sodium bisulfite was then used to analyze DNA single-strandedness at the single-molecule level, since this compound converts unpaired cytosines to uracils (and therefore to thymines) after polymerase chain reaction (PCR) amplification. We subjected the region-spanning markers IVS47 C3 to A4 (4 kb) to bisulfite analysis, and the same procedure was applied to the entire intron 71 (4.3 kb), which functioned as a control for background modification. In the IVS47 region and in IVS71, the overall cytosine conversion percentage was similar, and no evident association was observed between conversion occurrence and location of the DSB prone region or breakpoints. Similarly, the slightly increased frequency of consecutive cytosines (>7) in IVS47 compared to IVS71 did not warrant further speculation.
CONCLUSIONS AND SIGNIFICANCE
Duchenne muscular dystrophy, with an incidence of 1:3500 live male births, is one of the most common X-linked genetic diseases; the major dystrophin hot spot harbors 75% of deletion breakpoints involving the gene. Since identification of the hot spot, several attempts have been made to clarify the molecular determinants underlying deletion events. Early works, based on breakpoint mapping, reported that deletion breaks were scattered along the hot spot. More recent studies suggested that formation of DSBs represents the primary event in deletion formation.
Our results show that a good correlation exists between regions of the DMD locus that induce meiotic DSBs in yeast and the deletion breakpoints observed in human patients. If there is a hot spot for DSB formation and if DSBs are responsible for deletion events, we would expect a high concentration of deletion breakpoints near the hot spot. Indeed, cluster analysis indicated that the frequency of deletion breakpoints around the DSB prone region is significantly higher than would be expected from the hypothesis of random occurrence. Moreover, among 26 patients from an independent population, 11.5% of intron 47 deletion breakpoints were located within a 2 kb region centered around the DSB-prone region, resulting in a 3-fold higher breakpoint occurrence than expected by chance. These data strongly suggest that DMD deletion events with breakpoints in this interval are likely to arise via DSB formation.
Although several breakpoints have been detected in intron 44, no DSB was observed in our assay. It is possible that intron 44 has weak DSB sites that cannot be detected by our physical assay. Another possibility is that DSB sites reside outside the intron 44 sequence we tested.
The major dystrophin deletion hot spot covers 715 kb; in the commonly held view, breakpoint locations form a continuum along this huge region. Our data indicate that clustering of deletion breakpoints occurs within intron 47 (which harbors >10% of deletion breakpoints in the whole gene) and that one of the two clusters we identified maps to a region where DSBs occur during yeast meiosis. This finding suggests that DSBs mediate deletion formation in intron 47 and may account for the high frequency of meiotic recombination in the region. These results could indicate that the primary events in deletion formation occur within discrete regions and that the scattered breakpoint distribution reflects the variable degree of DSB end processing and the availability of a small (compared to the huge regions involved) deletion junction sample. It has been shown that clustering of recombination hot spots occurs in mammals over long genomic regions (37–39); we speculate that the DMD major hot spot represents one such region and that deletion and recombination hot spots can colocalize and have a common mode of origin (i.e., DSB formation).
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5635fje
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