Distinct properties of de novo mutations from whole genome sequencing of 50 patient-parent trios. M. Pinelli1,2, B. Tan3, M. van de Vorst1, R. Leach4, R. Klein4, L. E. L. Vissers1, H. G. Brunner1,5, J. A. Veltman1,5, A. Hoischen1, C. Gilissen1 1) Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; 2) 3Dipartimento di Medicina Molecolare e Biotecnologie Mediche, UniversitÓ Degli Studi di Napoli Federico II; 3) State Key Laboratory of Medical Genetics, Central South University, Changsha, China; 4) Complete Genomics Inc., Mountain View, CA, United States; 5) Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, Netherlands.

   De novo germline mutations create the genetic variability that is the driving force of species evolution, but also the cause of sporadic genetic diseases. It is therefore of great interest to study their occurrence and associated risk factors, such as the increased parental age. Whole Genome Sequencing (WGS) of parent-offspring trios allows for the first time to observe the mutational processes in a single generation in full detail. Here we report on the rate and pattern of De Novo Mutations (DNM) based on WGS of 50 trios at high (80x median) coverage. The trios consisted of patients with severe intellectual disability and their unaffected parents (age range at day of birth 20-39 years, mothers, 21-44, fathers). We identified 2815 private DNM, corresponding to an average of 56 per trio (range 32-84). Systematic validation of coding DNM resulted in an 80% validation rate. The single factor mostly associated with the number of identified DNM was the paternal age (p=10-6). By using the segregation of informative SNPs we determined the parental origin for 678 DNM (24%) and found a paternal/maternal ratio of 79% / 21%. Using the parental origin data we found a significant association between the paternal age and the number of paternal DNM (p=0.004, N=536) as well as a suggestive association between maternal age and the number of maternal DNM (p=0.05, N=142). By comparison to simulated mutations, we find that DNM do not occur completely random in the genome, but have sequence signatures enriched for CpG (observed=20.6%, expected=2.6%, p<10-16). Moreover a subset of the DNM were spatially clustered within individuals, lying within 10,000 nt proximity of eachother, (obs=1%, exp=0.0004%, p<10-16). These shower DNM (sDNM) are depleted of CpG dinucleotides (p<0.05), suggesting a less prevalent role of methylation-based mutagenesis. Moreover sDNM occur in regions that are enriched for common SNP (p<0.001), suggesting that they occur in mutation-prone regions. We validated 7 of these showers by Sanger sequencing and determined 2 to be paternally inherited and one maternally. These results suggest single mutational events for sDNM. We are currently re-sequencing all trios to obtain haplotype resolved genomes. We believe that these results provide insight into the mutational mechanisms of de novo mutations.

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