Decoding NF1 intragenic copy number changes. M. Hsiao1, A. Piotrowski1,2, T. Callens1, C. Fu1, L. Messiaen1 1) Department of Genetics, University of Alabama at Birmingham, Birmingham, AL; 2) Medical University of Gdansk, Gdansk, Poland.

   Genomic rearrangements are comprised of deletions, duplications, insertions, inversions and translocations causing both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements have been proposed such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR). However, to what extent these mechanisms contribute to rare, locus-specific pathogenic copy number changes (CNCs) remains unexplored. Furthermore, only a few studies resolved these pathogenic alterations at nucleotide-level resolution. Through array Comparative Genomic Hybridization (aCGH) as well as breakpoint-spanning PCR, we have identified the breakpoints and characterized the likely rearrangement mechanism of the NF1 intragenic CNCs in 76 unrelated subjects. Unlike the most typical recurrent rearrangement mediated by flanking low copy repeats (LCRs), NF1 intragenic CNCs vary in size and location. Microhomology from 1 to 41 bp was found in 57 patients (75% of 76 characterized breakpoint junctions), suggesting that the predominant mechanisms are DNA replication-based and microhomology-mediated. NAHR between repetitive elements was found in 16 individuals (21%). Additionally, Alu elements led to 15 Alu-Alu recombinations, and were also involved in 12 non-recurrent rearrangements including Alu insertion, NHEJ and FoSTeS/MMBIR. Hence, Alu elements were involved in as many as 27 unrelated patients (36%), suggesting their crucial role in generating NF1 intragenic CNCs. Furthermore, several Alu elements located in intron 2, 3, and 50, have demonstrated much stronger ability to mediate genomic rearrangements than other Alu elements. In addition to NAHR, NHEJ and FoSTeS/MMBIR, complicated rearrangements such as multiple NHEJ, multiple FoSTeS/MMBIR, serial replication slippage and Alu insertion were found in the NF1 intragenic CNCs. Furthermore, repetitive elements and non-B DNA structures, according to in silico analysis, were significantly associated with genomic rearrangements. We propose that these features might increase susceptibility to DNA double strand break or replication stalling. This locus-centered study based on a large set of breakpoint identifications provides important clues for NF1 molecular etiology, and also serves as a paradigm for other disorders involving genomic rearrangement.

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