Despite the potential of whole-genome sequencing (WGS) to improve patient diagnosis and care, the empirical value of WGS in the cancer genetics clinic is unknown. We performed WGS on members of two cohorts of cancer genetics patients: those with BRCA1/2 mutations (n = 176) and those without (n = 82). Initial analysis of potentially pathogenic variants (PPVs, defined as nonsynonymous variants with allele frequency < 1% in ESP6500) in 163 clinically-relevant genes suggested that WGS will provide useful clinical results. This is despite the fact that a majority of PPVs were novel missense variants likely to be classified as variants of unknown significance (VUS). Furthermore, previously reported pathogenic missense variants did not always associate with their predicted diseases in our patients. This suggests that the clinical use of WGS will require large-scale efforts to consolidate WGS and patient data to improve accuracy of interpretation of rare variants. While loss-of-function (LoF) variants represented only a small fraction of PPVs, WGS identified additional cancer risk LoF PPVs in patients with known BRCA1/2 mutations and led to cancer risk diagnoses in 21% of non-BRCA cancer genetics patients after expanding our analysis to 3209 ClinVar genes. These data illustrate how WGS can be used to improve our ability to discover patients’ cancer genetic risks.

Chronic myeloid leukemia (CML) and some acute lymphoblastic leukemias are characterized by the t(9;22) that encodes the BCR/ABL oncogene. Multiple mouse models of CML express BCR/ABL at high levels from non-Bcr promoters, resulting in the development of leukemias. In contrast, a significant fraction of healthy humans have been found to have BCR/ABL positive hematopoietic cells. To bridge the gap between the information derived from current mouse models and the non-leukemic humans with the BCR/ABL oncogene, we generated a knockin model with BCR/ABL p210 expressed from the Bcr locus. Unlike previous models, expression of BCR/ABL from the knockin allele did not induce leukemia. BCR/ABL mutant cells did exhibit favorable bone marrow engraftment compared to wild-type cells. These data suggest BCR/ABL expression alone is insufficient to induce disease. This new model allows for inducible spatial and temporal control of BCR/ABL expression for analysis of early steps in the pathogenesis of BCR/ABL-expressing leukemias.

Deficiency of huntingtin-interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated a deficiency allele where a floxed transcriptional stop cassette and a human cDNA were knocked into intron 1 of the mouse locus.-mediated germ line excision of the stop cassette resulted in expression of HIP1 and rescue of the knockout phenotype.-mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast, -mediated, brain-specific HIP1 expression did not rescue the phenotype. Metabolomics and microarrays of several knockout tissues identified low phosphocholine (PC) levels and low glycerophosphodiester phosphodiesterase domain containing 3 (Gdpd3) gene expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC, downregulation could lead to the low PC levels. To test whether contributes to the deficiency phenotype, we generated knockout mice. Double knockout of and worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More-detailed knowledge of how deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.

Myeloproliferative neoplasms (MPNs) such as chronic myelogenous (CML) and chronic myelomonocytic leukemias (CMML) are frequently induced by tyrosine kinase oncogenes. Although these MPNs are sensitive to tyrosine kinase inhibitors such as imatinib, patients often relapse upon withdrawal of therapy. We used a model of MPN, which is induced by co-expression of the oncoproteins HIP1/PDGFβR (H/P) and AML1/ETO (A/E) from their endogenous loci, to examine the mechanisms of disease development and recurrence following imatinib withdrawal. Although the MPN displayed a full hematologic response to imatinib, 100% of the diseased mice relapsed upon drug withdrawal. MPN persistence was not due to imatinib resistance mutations in the H/P oncogene or massive gene expression changes. Within one week of imatinib treatment, more than 98% of gene expression changes induced by the oncogenes in isolated hematopoietic stem and progenitor cells (LSKs) normalized. Supplementation of imatinib with G-CSF or arsenic trioxide reduced MPN-initiating cell frequencies and the combination of imatinib with arsenic trioxide cured a large fraction of mice with MPNs. In contrast, no mice in the imatinib-treated control cohorts were cured. These data suggest that treatment with a combination of arsenic trioxide and imatinib can eliminate refractory MPN-initiating cells and reduce disease relapse.

47Deficiency of huntingtin interacting protein 1 (Hip1) results in degenerative phenotypes. 48Here we generated a Hip1 deficiency allele where a floxed transcriptional stop-cassette 49 and a human HIP1 cDNA were knocked-in to intron 1 of mouse Hip1 locus. CMV-Cre-50 mediated germline excision of the stop-cassette resulted in expression of HIP1 and rescue 51 of the Hip1 knockout phenotype. Mx1-Cre-mediated excision led to HIP1 expression in 52 spleen, kidney and liver, and also rescued the phenotype. In contrast, GFAP-Cre-53 mediated HIP1 expression in brain did not rescue the phenotype. Metabolomics and 54 microarrays of several Hip1 knockout tissues identified low phosphocholine (PC) levels 55 and low Glycerophosphodiester Phosphodiesterase Domain Containing 3 (Gdpd3) 56 expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of 57 choline, a precursor of PC, Gdpd3 downregulation could lead to the low PC levels. To 58 test if Gdpd3 contributes to the Hip1 deficiency phenotype, we generated Gdpd3 59 knockout mice. Double knockout of Gdpd3 and Hip1 worsened the Hip1 phenotype. This 60 suggests that Gdpd3 compensates for Hip1 loss. More detailed knowledge of how Hip1 61 deficiency leads to low PC will improve our understanding of HIP1 in choline 62 metabolism in normal and disease states. 63 64 65 amplified from pcDNA3-hHip1 plasmid. The IRES (~0.6 kb) and eGFP (~0.9 kb) 128 sequences were amplified from pIRES2-AcGFP1 and pEGFP-N1 plasmid, respectively. 129The final vector was obtained by standard molecular cloning. Aside from the 130 homology arms, the final vector also contains a Lox-Stop-Lox cassette (~1.5 kb), mHip1 131 partial intron1+ partial exon 2, hHip1 cDNA, IRES, EGFP + polyA, Frt sequences flanking 132 the Neo expression cassette (the neo cassette was used for positive selection of the 133 electroporated ES cells), and a DTA expression cassette (for negative selection of the ES 134