Discrete Paralogous Recombination Sites

Despite 51 kb of 97.5% sequence identity between NF1-REP-P1-51 and NF1-REP-M-51, paralogous recombination occurred preferentially at two discrete sites. NF1 microdeletion breakpoints were mapped at the nucleotide level to intervals defined by paralogous sequence variants (PSV; also known as NF1-REP-specific variants). The product of paralogous recombination is a chimeric NF1-REP-P1/NF1-REP-M and shows a pattern of PSVs with transition from NF1-REP-P1 PSVs to NF1-REP-M PSVs at the breakpoint interval. Breakpoint mapping was facilitated by use of human/rodent somatic hybrid cell lines that carried only the deleted homolog 17 of the patient (17,23). Sixty-nine percent (N = 78) of NF1 microdeletion cases had breakpoints that clustered at paralogous recombination sites 1 and 2 (PRS1 and PRS2) (Fig. 3) (17,23,24) (23). PRS2 harbored 51% of breakpoints, whereas PRS1 harbored 18%. A single case UWA160-1 had a distinct breakpoint centromeric to PRS sites (Fig. 3). The PRS1 and PRS2 regions are 4.1 and 6.3 kb in length, respectively, and are 14.5 kb apart. Each PRS has a hotspot where the majority of breakpoints mapped; PRS2 has a 2.3-kb hotspot that accounts for 93% of breakpoints, whereas PRS1 has a 0.5-kb hotspot accounting for 60% of breakpoints. During sequence analysis of recombinant PRS in several cases, instead of a perfect transition of PSVs from NF1-REP-P1 to -M, the PSVs were in "patches" with a complex transition from NF1-REP-P1 to -M to -P1 to -M (17,23), indicating apparent gene conversion events. These regions were relatively short (<627 bp) and, like similar events of REP-mediated rearrangements in CMT1A and AZFa and IDS (28-31), are considered consistent with a mechanism of double-strand break repair.

There was no significant difference between PRS1- and PRS2-mediated microdeletions for the parent of origin or for de novo vs familial cases (17,23). In a series of 59 NF1 microdeletion cases for which clinical evaluation of the parents was available, 10% inherited the disease, and presumably the microdeletion, from an affected parent. Among 45 de novo cases where parental origin could be determined, 80% were of maternal origin. The recent development of deletion-specific amplification assays that detect the recurrent NF1 microdeletions at PRS1 and PRS2 will facilitate the assembly of patient cohorts of the same genotype for clinical evaluation and will quickly identify those patients with variant deletions, which will be important to narrow the critical region of the deletion responsible for the increased tumor load and malignancy risk of microdeletion patients (17,23).

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