High resolution FISH analysis of NF1 REP and non-REP microdeletions evidences nonhomologous end joining mediated rearrangements

NF1 microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and its flanking regions. NF1 patients carrying gross deletion, often displaying complex phenotype, account for 5-10% of all NF1 mutations. Most of the NF1 deletions (80%) originate by unequal homologous recombination of repeated sequences (REP-P and M) mapped to 17q11.2, while the remaining show unusual breakpoints. We performed high-resolution FISH analysis of 20 NF1 microdeleted patients with the aim of mapping non recurrent deletion breakpoints and verifying the presence of other recombination-prone genome architectural motifs. Conventional FISH analysis allowed us to identify 17 REP-deleted patients, and three patients carrying atypical deletions of 1.3 Mb, 1.5 Mb and 3 Mb respectively. By fiber-FISH, we identified genomic intervals of ~100 Kb comprising the breakpoint regions, which proved to be affected by reported deletions and translocation. The generation of several locus-specific FISH probes allowed us to restrict the critical deletion endpoint intervals to a few Kb. This approach led us to hypothesize that small blocks of REPs cluster around the 1.3 Mb deletion breakpoints and LPA1 repeats may be involved in the 1.5 Mb deletion breakpoints. The precise restriction of the deletion interval, by locus-specific probe, allowed us to isolate the 3 Mb deletion junction fragment by long-range PCR. Sequencing of the novel junction fragment indicated that the deletion is likely caused by non homologous end joining. We also established the exact deletion gene content in non-REP deletions which, by comparison with the REP deletion gene content, will contribute to carrying out genotype-phenotype correlation studies