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// Nardin Samuel 1, 2, 3 , Gavin Wilson 3, 4 , Badr Id Said 2 , Anna Pan 2 , Genevieve Deblois 5 , Nicholas W. Fischer 6 , Roumiana Alexandrova 7 , Guillermo Casallo 7 , Tara Paton 7 , Mathieu Lupien 5 , Jean Gariepy 6 , Daniele Merico 7 , Thomas J. Hudson 1, 3, 4 , David Malkin 1, 2, 8 1 Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Canada 2 Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Canada 3 Ontario Institute for Cancer Research, Toronto, Canada 4 Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, Canada 5 Princess Margaret Cancer Centre, University Health Network, Toronto, Canada 6 Department of Physical Sciences, Sunnybrook Research Institute, Toronto, Canada 7 The Centre for Applied Genomics (TCAG), Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada 8 Division of Hematology/Oncology, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, Canada Correspondence to: David Malkin, email: david.malkin@sickkids.ca Keywords: miR-34A, p53, TP53, cell cycle, non-coding RNA Received: April 07, 2016 Accepted: May 19, 2016 Published: July 06, 2016 ABSTRACT microRNA-34A is a critical component of the p53 network and expression of miR- 34A is down-regulated by promoter hypermethylation or focal deletions in numerous human cancers. Although miR-34A deregulation may be an important driver in cancer, the endogenous role of this microRNA in cellular homeostasis is not well characterized. To address this knowledge gap, we aimed to determine the transcriptional landscape of the miR-34A-p53 axis in non-transformed cells. Using primary skin-derived fibroblast cell lines from patients who developed childhood cancers, and who harbor either germline TP53 mutations or are TP53 wild type, we sought to characterize the transcriptional response to miR-34A modulation. Through transcriptome-wide RNA-Sequencing, we show for the first time that in human non- transformed cells harboring TP53 mutations, miR-34A functions in a noncanonical manner to influence noncoding RNA networks, including RNA components of the minor (U12) spliceosome, as well as TP53 -dependent and independent epigenetic pathways. miR- 34A-regulated transcripts include known cell cycle mediators and abrogation of miR-34A leads to a TP53 -dependent increase in the fraction of cells in G2/M. Collectively, these results provide a framework for understanding the endogenous role of the miR-34A signaling axis and identify novel transcripts and pathways regulated by the essential miR-34A-p53 tumor suppressor network.

Purpose Hemiplegia is a subtype of cerebral palsy (CP) in which one side of the body is affected. Our earlier study of unselected children with CP demonstrated de novo and clinically relevant rare inherited genomic copy-number variations (CNVs) in 9.6% of participants. Here, we examined the prevalence and types of CNVs specifically in hemiplegic CP. Methods We genotyped 97 unrelated probands with hemiplegic CP and their parents. We compared their CNVs to those of 10,851 population controls, in order to identify rare CNVs (

Background Epstein–Barr virus (EBV) is associated with several diseases, including infectious mononucleosis (IM) and malignant disorders, including post-transplant lymphoproliferative disorder (PTLD). The relationship between strains of the virus and disease manifestations or illness severity is of interest. Such strains have been defined by genetic variations in the major viral genes. Data involving the patterns of genetic diversity of the virus in different populations are required. We examined the genetic diversity of the BZLF1 gene, which is a major lytic gene of the virus. Methods We sequenced the BZLF1 gene of EBV following amplification from DNA that was extracted from blood obtained from pediatric bone marrow transplant (BMT) patients and children and young adults with IM. Sequencing was done by Sanger methodology (dideoxy DNA sequencing) and the sequences were aligned with a reference strain of EBV using Geneious software. The variant burden and types of single nucleotide variants (SNV) were compared across the 3 exons of the BZLF1 gene. Results We sequenced the BZLF1 gene using 21 patients with IM (median age 14, age range 2–19 years) and 11 who underwent bone marrow transplantation (median age 6, range 3–13 years). Three of 11 BMT patients developed post-transplant lymphoproliferative disorder (PTLD). Among the 3 exons, exon 1 had the greatest diversity across both study groups. There was a tendency for less diversity among PTLD samples, with no sample containing >1 single nucleotide variant (SNV) in contrast to the other samples. In samples that contained SNVs, there was a non-statistically significant trend for more SNVs to occur among the IM samples compared with PTLD samples (median 4.5 and 0, respectively; P > 0.05). Additionally, 2/11 (18.2%) BMT sequences contained more than 1 SNV compared with 7/21 (33.3%) IM sequences (P > 0.05). Conclusion There was a tendency for more genetic diversity among samples from patients with IM compared with bone marrow transplant patients, notably those with PTLD. Further studies will determine if this tendency is due to selective pressures in the transplant setting, including but not limited to the use of antiviral agents directed at the lytic phase of EBV. Disclosures All authors: No reported disclosures.

Background The Epstein–Barr virus (EBV) is associated with several diseases, including infectious mononucleosis as well as malignant disorders. The relationship between strains of the virus and disease manifestation or illness severity is of interest. Such strains have been defined by genetic variations in the major viral genes. As a first step toward a better understanding of the relationship between strains and clinical outcomes, data are required on the patterns of genetic diversity of the virus in different populations. In this study, we examined the genetic diversity of the BZLF-1 gene, which is a major lytic gene of the virus. Methods We sequenced the BZLF-1 gene of EBV following amplification from DNA that was extracted from blood and saliva from previously healthy Canadian children and young adults with infectious mononucleosis. Sequencing was done by Sanger methodology (dideoxy DNA sequencing) and the sequences were aligned with a reference strain of EBV using Geneious software. The variant burden and types of single nucleotide variants were compared in blood and saliva samples. Results Twenty-six samples were obtained from 24 patients less than 24 years of age (16 saliva and 10 blood samples). Two subjects provided paired blood and saliva samples at the same visit. Among 36 single nucleotide variations (SNVs), 22% were common to both blood and saliva samples. There was a nonstatistically significant trend for more SNVs among blood compared with saliva samples (median 6 and 1, ranges 0–8 and 0–9, respectively). Of the 3 exons of BZLF-1, exon 1 had the greatest frequency of SNVs compared with exons 2 and 3. Among the paired samples of blood and saliva, there were different genetic variants of the BZLF-1 gene in the blood compared with the saliva samples obtained from patients with infectious mononucleosis. Conclusion Among patients with infectious mononucleosis, different genetic variants of EBV may be present in blood compared with saliva. Blood samples revealed viral strains with a tendency for more genetic diversity compared with saliva. The potential compartmentalization of strains is of relevance in sample selection for the evaluation of the potential clinical impact of the genetic diversity of EBV. In addition, the potential impact on disease pathogenesis is of interest. Disclosures All authors: No reported disclosures.