Whole Genome Sequencing of two individuals with excessive numbers of de novo CNVs. P. Liu1, K. Walter2, A. Wuster2, T. Gambin1, V. Gelowani1, K. Writzl3, S. Lindsay2, C. Carvalho1, M. Withers1, C. Gonzaga1, J. Wiszniewska1, A. Patel1, B. Rautenstrauss4, R. Gibbs1, M. Hurles2, J. Lupski1 1) Molec & Human Gen, Baylor College Med, Houston, TX; 2) Wellcome Trust Sanger Institute, Hinxton, UK; 3) Institute of Medical Genetics, UMC, Ljubljana, Slovenia; 4) Medical Genetics Center, Bayerstrasse 3-5, Munich, Germany.

   Large de novo copy number variation (dnCNV) was estimated to occur constitutionally about once in every 100 individuals. It is extremely rare to observe individual genomes with two or more independently generated dnCNVs. Here we describe two subjects with excessive numbers of dnCNVs. They were referred to clinic because of developmental problems and multiple congenital anomalies. Genome-wide and region-specific array CGH analyses identified eight (50kb to 6.4Mb) and ten (210kb to 4.7Mb) dnCNVs in the two subjects, respectively. In order to characterize the full spectrum of dnCNVs and to glean insights into mechanism, whole genomes of the two trios were sequenced by Illumina Technology to an average depth of sequence coverage of 25X. Two CNV callers, BreakDancer and Pindel, were used to identify new dnCNVs or complexities not detected by aCGH, and to help obtain breakpoint sequences. All the dnCNVs identified from WGS analysis only were in proximity to the large dnCNVs originally revealed by arrays, suggesting that they likely belong to individual complex genomic rearrangements, and that the spectrum of dnCNVs in these two subjects does not seems to comprise smaller sizes. In addition, duplications in both subjects appear in the form of tandem duplication as well as insertional translocation. In a few loci, breakpoint complexities and triplications were identified, suggesting that the rearrangements were likely produced by replication mechanisms. Using variants specific to each parent, haplotypes were phased in patient genomes. In both patients, the dnCNVs were a mixture of paternal and maternal events, consistent with postzygotic timing of the CNV occurrence. Interestingly, in one of the subjects, the vast majority of dnCNVs was derived from the maternal chromosomes. In the same patient, de novo point mutations preferentially occurred in the paternal chromosome, may be reflecting advanced paternal age. The genomic distribution of de novo point mutations are being examined and validated in a comprehensive manner to investigate potential association with sites of dnCNV. Our results document a genome-wide spectrum of dnCNVs in rare cases of subjects with excessive number of mutations, and we suggest that errors in the cellular DNA replication machinery could lead to a propensity for long distance template switching events and multiple independent de novo rearrangements. Our findings have important implications for genomic disorders, cancer and evolution.