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Fig. 3. Genomic context of the paralogous recombination hotspots for NF1 microdeletion. (A) Alignment, identity, and sequence features of the 51-kb high identity NF1-REP-P1-51 and NF1-REP-M-51 paralogs. Alignment mismatch panel shows gap alignments ranging from 1 to 50 bp between NF1-REP-P1-51 and NF1-REP-M-51 in BACs RP11-271K11 and RP11-640N20, respectively. PSVs for each REP, indels excluded, with a sliding 100-bp window are shown, including a matrix attachment site (MAR), an apparent gene conversion tract with variants matching NF1-REP-E19, and a 700-bp segment of perfect match with statistical evidence of gene conversion. The positions of promoter like sequences not associated with known genes are indicated in the lower panel. (B) Detailed structure of the PRS2 region. Breakpoint intervals are shown along with the 2.3-kb hotspot, which harbors 93% of breakpoint intervals in PRS2 region. Finer localization of the 700-bp gene conversion tract and the promoter like sequences from (A) are shown. Nucleotide positions for both panels refer to the NF1-REP-P1-51 in BAC RP11-271K11. (Adapted from ref. 24 with permission.)

Basic Local Alignment Search Tool (BLAST; www.ncbi.nlm.nib.gov/blast) comparison of PRS1 and PRS showed no significant sequence identity with the exception of Alu elements, LINES and other high-copy repeats, which typically shared less than 80% identity in short segments (24). PRS1 and 2 regions have quite different patterns of G+C content; the PRS2 hotspot is very G+C rich, while the PRS1 hotspot is not (Fig. 3). Both PRS hotspots are 1-2 kb distal to relative large alignment gaps (Fig. 3A), yet these did not suppress pairing as recombination in at least a few cases occurred within less than 1 kb from the gaps. The PRS regions are not of greater or lesser paralogous sequence identity as shown by the spatial distribution of PSVs, which are relatively evenly distributed across the NF1-REP-51 segment (Fig. 3) (24). Numerous tests for the presence of motifs with demonstrated or suspected roles in recombination, transcription, or translation were performed. A Chi element within PRS2 was identified (17), but this association is not preferential, as it is one of four evenly spaced Chi elements in NF1-REP-51 (24). Figure 3B shows the location of a CpG island and two promoter-like sequences, which although not associated with any known gene, may function to provide chromatin accessibility. Statistical tests for gene conversion identified the 700-bp perfect match between NF1-REP-P1-51 and NF1-REP-M-51 that coincided with PRS2 (Fig. 3B). Although perfect sequence match may contribute to breakpoint localization, these results suggest that perfect tracts at paralogous recombination hotspots may be a result of gene conversion at sites at which preferential pairing occurs for other unknown reasons (24). A search for palindromes, which are associated with other genomic rearrangements (32-35), found no palindromes larger than 18 bp and separated by 63 bp. The palindromes within the KIAA0563-y of NF1-REP-P1 are considered too distant to influence recombination at the PRS regions (Fig. 2A).

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