Next-generation sequencing of duplication CNVs reveals that most are tandem and some disrupt genes at breakpoints. K. Rudd, K. E. Hermetz, B. Weckselblatt, S. Newman Dept Human Gen, Emory Univ Sch Med, Atlanta, GA.

   Copy number variation (CNV) in the form of large deletions and duplications is a major cause of neurodevelopmental disorders. Though duplications are a common finding in cytogenetic testing, the clinical consequences of duplication CNVs are particularly difficult to interpret because the genomic structure and precise breakpoints are unknown. Duplications are usually less deleterious than deletions, and could lead to disease through gene triplosensitivity, gene fusion, and/or gene disruption. We fine-mapped clinically relevant duplications in 189 subjects referred for cytogenetic testing at Emory Genetics Laboratory. Using sequence capture, massively parallel paired-end sequencing, and confirmatory Sanger sequencing, we sequenced breakpoints of 90 non-recurrent duplications to the basepair. These large duplications (27 kb- 25 Mb; median 600 kb) exist as tandem duplications (87%), insertional translocations (3%), or complex rearrangements (10%). We identified two major classes of complex duplications, those that connect two adjacent duplications (dup-dup) and those with triplications embedded in duplications (dup-trip). Sequencing breakpoint junctions revealed that both dup-dup and dup-trip structures can be tandem or inverted. Though duplicated genes may be disrupted by breakpoints, most duplications retain intact copies of genes at duplication boundaries. Few duplication junctions are predicted to result in novel isoforms of single genes (2%) or in-frame fusions of two different genes (11%). One of the three insertional translocations disrupted a gene at the insertion site and is predicted to result in an in-frame fusion of exons 1-3 of USP20 and exons 3-45 of COL4A6. Intragenic duplications of CNTN4 and TCOF1 disrupted reading frames, while duplications within OPCML, DMD, and PAFAH1B1 were completely intronic. We detected a 324-kb intergenic duplication that fuses exons 1-6 of SOS1 and exons 2-33 of MAP4K3 in-frame in a child with some features of Noonan syndrome. Our large-scale analysis of genomic gains showed that most duplications are local and tandem. Sequence analysis of duplications revealed genomic structures (triplications, inversions) and gene alterations (disruptions, fusions) that are not detectable by clinical array testing alone. These data are essential to interpret the phenotypic impact of duplications in the human genome.

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