ABSTRACT: Genomic rearrangements may cause 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 gene-specific pathogenic copy-number changes (CNCs) remains understudied. Furthermore, only few studies resolved these pathogenic alterations at nucleotide-level resolution. Accordingly, our aim is to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array Comparative Genomic Hybridization (aCGH), we have identified the breakpoints and characterized the likely rearrangement mechanism of the NF1 intragenic CNCs in 78 unrelated patients. Unlike the most typical recurrent rearrangements mediated by flanking low copy repeats (LCRs), NF1 intragenic CNCs have diverse breakpoint locations, and are characterized by different rearrangement mechanisms. We propose the DNA replication-based mechanisms comprising FoSTeS/MMBIR and serial replication stalling to be the predominant mechanism leading to NF1 intragenic CNCs. In addition to the loop of a 197-bp palindrome located in intron 40, four Alu elements located in intron 1, 2, 3 and 50 were also identified as significant intragenic rearrangement hotspots within the NF1 gene. However, no clear genotype-phenotype correlations could be identified among the NF1 patients carrying NF1 intragenic CNCs. Patient DNA samples with non-overlapping CNCs, as estimated by MLPA, were labeled with Cy3 and Cy5 fluorophores respectively, and hybridized onto the microarray. Alternatively, patient DNA was hybridized versus unrelated individual blood DNA. No hybridizations using biological replicates were performed. In total 6 samples were included. Please note that our experimental setup included a hybridization in which two DNA samples with non-overlapping deletions, from two NF1 patients, were hybridized in one experiment (sample codes: 1253 and 1403). In this specific case, assigning test or reference function to the samples was a matter of arbitrary choice. However, if needed, sample 1253 can be denoted as test, and sample 1403 can be denoted as reference in this experiment.
