A region in the dystrophin gene major hot spot harbors a cluster of deletion breakpoints and generates double‐strand breaks in yeast

Abstract

Deletions within the dystrophin gene (DMD) account for >70% of mutations leading to Duchenne and Becker muscular dystrophies (DMD and BMD). Deletion breakpoints were reported to be scattered within regions that also represent meiotic recombination hot spots. Recent studies indicates that deletion junctions arise from nonhomologous end joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammals. Here we show that a region in intron 47 (i.e., a major deletion hot spot in the DMD gene) generates DSBs during meiosis in yeast and harbors a cluster of previously sequenced deletion breaks. Mapping of breakpoints in 26 BMD/DMD patients indicated that the frequency of breakpoint occurrence around this region is 3-fold higher than expected by chance. These findings suggest that DSBs mediate deletion formation in intron 47 and possibly account for the high frequency of meiotic recombination in the region. Statistical analysis indicated the presence of at least one other breakpoint cluster in intron 47. Taken together, these results suggest that the primary events in deletion formation occur within discrete regions and that the scattered breakpoint distribution reflects both a variable degree of DSB end processing and the availability of a small (compared to the huge regions involved) deletion junction sample.—Sironi, M., Pozzoli, U., Comi, G. P., Riva, S., Bordoni, A., Bresolin, N., Nag, D. K. A region in the dystrophin gene major hot spot harbors a cluster of deletion breakpoints and generates double-strand breaks in yeast. mutations in the dystrophin gene (DMD) located on Xp21 are responsible for Duchenne and Becker muscular dystrophies (DMD and BMD, respectively). The gene, composed of 79 exons, spans >2.5 Mb and occupies ∼0.1% of the genome (1⤻ ,2)⤻ . Worldwide incidence of BMD is ∼1/30,000 male births whereas occurrence of DMD is 1 in 3500, with one-third of the cases arising from new mutations (3)⤻ . Large intragenic deletions and duplications together account for >two-thirds of the mutations leading to DMD and BMD and, despite heterogeneity in both deletion size and location, two hot spots have been identified: a minor hot spot is located around intron 7, while the major one involves exons 40–55 (4⤻ ,5)⤻ . These two large gene regions also represent major meiotic recombination hot spots (6)⤻ . Sequencing of deletion junctions involving both hot spots revealed no clear clustering of chromosome breaks and failed to identify any element or feature that could account for deletion formation (7⤻ 8⤻ 9⤻ 10)⤻ . Analysis of available deletion junctions also indicated that they arise from rejoining of broken ends via nonhomologous end joining (NHEJ) (8⤻ 9⤻ 10)⤻ . NHEJ plays a central role in processes of illegitimate recombination in mammals (reviewed in ref. 11⤻ ) and represents a major pathway for the repair of DNA double-strand breaks (DSBs) (11). These observations suggest that the primary event leading to dystrophin deletions could be DSB formation within the DMD locus. DSBs are initiators of several genetically programmed events, such as V(D)J recombination and a high level of meiotic exchange (12⤻ 13⤻ 14)⤻ ; however, DSBs also arise during DNA replication and due to other endogenous processes. In the yeast Saccharomyces cerevisiae, formation of meiosis-specific DSBs has been studied extensively both genetically and physically; DSBs generally occur at specific locations, the so-called recombination hot spots (reviewed in ref. 15⤻ ). Yeast artificial chromosomes (YACs) containing human genomic DNA, covering portions of the DMD gene, undergo DSB formation during meiosis in yeast, and the frequency of YAC double-strand breakage reflects the degree of meiotic recombination in humans (16)⤻ . These observations suggest that sequence or structural features that contribute to DMD genomic instability and DSB formation are preserved in yeast and represent target sites for meiotic DSBs. We have suggested (10)⤻ that intervals with higher breaking frequency within already described deletion-prone regions could exist in the dystrophin gene. In particular, upon analysis of deletion breakpoints in a population of BMD patients, we identified a 1250 bp interval in intron 47 (IVS47) where three deletion breakpoints were located; given the size of IVS47 (54 kb), the close proximity of these breaking sites can hardly be due to chance. We therefore designed experiments to investigate whether the region encompassing the three IVS47 breakpoints also generates DSBs during yeast meiosis. Several other deletion breakpoints have been identified and sequenced within the large intron 44 (IVS44, 148 kb). To determine whether they also represent DSB sites, we included three intron 44 regions in our studies. Our data show that a region in IVS47 induces DSBs during meiosis in yeast and harbors a breakpoint cluster, suggesting that deletion events arise via a DSB repair pathway. MATERIALS AND METHODS Yeast strains, media, and genetic techniques Saccharomyces cerevisiae strains were derived from AS4 (α trp1–1 arg4–17 tyr7–1 ura3 ade6) and AS13 (α leu2-Bst ura3–52 ade6) background (17)⤻ . All his4-DMD mutant alleles were introduced into the AS13 chromosome by a two-step replacement procedure. The rad50S mutation was introduced into the chromosome by use of the plasmid pNKY349, as described previously (18)⤻ . Haploid and diploid strains used in this study were as follows: DNY107 (AS4, rad50S); DNY115 (AS13, rad50S X DNY107); MSY11 (AS13 his4-DMD71 X DNY107); MSY12 (AS13 his4-DMD47–1 X DNY107); MSY13 (AS13 his4-DMD47–2 X DNY107); MSY14 (AS13 his4-DMD47–3 X...