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Keywords: Chromosomal abnormalities; Chromosomal structure/function; Copy number/structural variation; Genomics; Massively parallel sequencing

C. Carvalho 1; C. Beck 1; Z. Akdemir 1; F. Sedlazeck 2; Q. Meng 2; J. Hu 2; H. Doddapaneni 2; Z. Chong 3; E. Chen 1; M. Withers 1; S. Jhangiani 2; A. English 2; Y. Han 2; K. Chen 4; D. Muzni 2; R. Gibbs 2; C. Shaw 1; P.J. Hastings 1; J.R. Lupski 1,2

1) Dept Molecular Human Genetics, Baylor Col Medicine, Houston, Texas.; 2) Human Genome Sequencing Center, BCM, Houston TX; 3) Department of Genetics and the Informatics Institute, the University of Alabama at Birmingham, Birmingham, AL; 4) The University of Texas MD Anderson Cancer Center, Houston, TX

Copy number variation (CNV), including duplications and triplications formed by microhomology-mediated break-induced replication (MMBIR) has been shown to be generated mainly by intrachromosomal events, i.e. only segments originating from the same homologue are part of the end product. The hypothesized reason is that template-switches (TS) within the same chromosome are thought to be facilitated by the physical proximity in the nucleus due to linkage as well as to cis intrachromosomal interactions. Autosomal triplications followed by runs of homozygosity (ROH) are clinically relevant exceptions: they are generated post-zygotically due to interhomolog TS which can lead to imprinting perturbations and associated diseases. To gain mechanistic insights into the formation of de novo autosomal copy number gains and complex rearrangements, we studied 11 trios (patient + parents) in which probands were referred with congenital malformations carrying rearrangements involving 17p11.2. Trios were studied by combining array comparative genomic hybridization (aCGH), long-and short-reads DNA sequencing technologies including a targeted 7Mb Illumina short read spanning the 17p interval and Pacific Biosciences SMRT sequencing approach. Phasing of the copy number gains were obtained by B-allele frequency analysis provided by trio joint-calling, whereas array data and PacBio were used to resolve CNV breakpoint junctions. Intrachromosomal rearrangements were observed in 6 out of 11 cases and interchromosomal events in 1 out of 11, confirming that a majority of events occurred within the same homolog. Surprisingly, though, in 4 out of 11 (36%) cases the CNVs constitute a mix of both intra- and inter-homologue amplified segments rather than originating from only one ancestral chromosome, all of them formed by pre-zygotic mutagenesis. Intriguingly, only a subgroup of the template-switching events led to CNVs, similar to what was previously observed for post-zygotic autosomal triplications. Our results indicate that high-resolution B-allele frequency analysis is a helpful tool to uncover CNV formation and reveal TS events that can result in increased rearrangement complexity. Importantly, DNA segments originating from two distinct parental chromosomes contributing to the formation of the same rearrangement strongly support a TS model of generating amplifications and have further implications for human diseases, including those resulting from perturbations of imprinting.