Sequencing of unbalanced translocation junctions reveals mutational mechanisms and gene fusions. B. Weckselblatt, M. K. Rudd Human Genetics, Emory University, Atlanta, GA.

   Unbalanced chromosome translocations are a major contributor to intellectual disability and other neurodevelopmental disorders. Most translocation breakpoints are not recurrent and the mechanisms of translocation formation are largely unknown. Sequencing translocation breakpoints can point to specific types of double-strand break repair and reveal breakage-susceptible regions of the genome. In addition, sequence analysis of breakpoint junctions can reveal more complex rearrangement structures than expected from copy number studies alone. In a cohort of 55 human subjects with unique unbalanced translocations, we are characterizing chromosome breakpoints using a combination of array comparative genome hybridization, targeted sequence capture, and next-generation sequencing. Of the 33 sequenced translocations, 22 have between zero and four basepairs (bp) of microhomology at the breakpoint junction, consistent with nonhomologous end joining (NHEJ) or microhomology-mediated break-induced replication (MMBIR). Four junctions had 450-3023 bp of sequence homology typical of nonallelic homologous recombination (NAHR) between interspersed repeats. Notably, seven translocations had short inserted sequences at the junctions, suggesting a DNA replication-based mechanism of repair. Mapped insertions originate from sequence nearby the junction or up to 56 kilobases away from the translocation breakpoint. A recent study of 12 unbalanced translocation breakpoints suggested that the primary mechanism of rearrangement was NAHR. However, our sequence analysis of unbalanced translocations junctions revealed NHEJ/MMBIR as the dominant form of repair, which is consistent with other large-scale studies of copy number variation (CNV) breakpoints. Ongoing studies include sequence analysis of translocations between three, four, or five different chromosomes that may arise via chromothripsis. Junction sequencing has also uncovered two potential novel fusion genes, FSTL5-PRDM16 and SMOC2-PROX1. In both translocations, disrupted genes are in the same direction, breakpoints lie within introns, and the resulting fusion is predicted to be in-frame. As in cancer genomes, fusion genes can lead to gain-of-function and contribute to disease. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of molecular mechanisms of translocations.

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