No abstract

Dyslipidemia is a well-established risk factor for cardiovascular diseases. Although, advances in genome-wide technologies have enabled the discovery of hundreds of genes associated with blood lipid phenotypes, most of the heritability remains unexplained. Here we performed targeted resequencing of 13 bona fide candidate genes of dyslipidemia to identify the underlying biological functions. We sequenced 940 Sikh subjects with extreme serum levels of hypertriglyceridemia (HTG) and 2,355 subjects were used for replication studies; all 3,295 participants were part of the Asian Indians Diabetic Heart Study. Gene-centric analysis revealed burden of variants for increasing HTG risk in GCKR (p = 2.1x10 -5 ), LPL (p = 1.6x10 -3 ) and MLXIPL (p = 1.6x10 -2 ) genes. Of these, three missense and damaging variants within GCKR were further examined for functional consequences in vivo using a transgenic zebrafish model. All three mutations were South Asian population-specific and were largely absent in other multiethnic populations of Exome Aggregation Consortium. We built different transgenic models of human GCKR with and without mutations and analyzed the effects of dietary changes in vivo . Despite the short-term of feeding, profound phenotypic changes were apparent in hepatocyte histology and fat deposition associated with increased expression of GCKR in response to a high fat diet (HFD). Liver histology of the GCKR mut showed severe fatty metamorphosis which correlated with ~7 fold increase in the mRNA expression in the GCKR mut fish even in the absence of a high fat diet. These findings suggest that functionally disruptive GCKR variants not only increase the risk of HTG but may enhance ectopic lipid/fat storage defects in absence of obesity and HFD. To our knowledge, this is the first transgenic zebrafish model of a putative human disease gene built to accurately assess the influence of genetic changes and their phenotypic consequences in vivo .

Dyslipidemia is a well-established risk factor for cardiovascular diseases. Although, advances in genome-wide technologies have enabled the discovery of hundreds of genes associated with blood lipid phenotypes, most of the heritability remains unexplained. Here we performed targeted resequencing of 13 bona fide candidate genes of dyslipidemia to identify the underlying biological functions. We sequenced 940 Sikh subjects with extreme serum levels of hypertriglyceridemia (HTG) and 2,355 subjects were used for replication studies; all 3,295 participants were part of the Asian Indians Diabetic Heart Study. Gene-centric analysis revealed a burden of variants for increasing HTG risk in GCKR (p=2.1×10−5), LPL (p=1.6×10−3) and MLXIPL (p=1.6×10−2) genes. Of these, three missense and damaging variants within GCKR were further examined for functional consequences in vivo using a transgenic zebrafish model. All three mutations were South Asian population-specific and were largely absent in other multiethnic populations of the Exome Aggregation Consortium. We built different transgenic models of human GCKR with and without mutations and analyzed the effects of dietary changes in vivo. Despite the short-term feeding, profound phenotypic changes were apparent in hepatocyte histology and fat deposition associated with increased expression of GCKR in response to a high fat diet (HFD). Liver histology of the GCKRmut showed severe fatty metamorphosis which correlated with ~7 fold increase in the mRNA expression in the GCKRmut fish even in the absence of a high fat diet. These findings suggest that functionally disruptive GCKR variants not only increase the risk of HTG but may enhance ectopic lipid/fat storage defects in the absence of obesity and HFD. To our knowledge, this is the first transgenic zebrafish model of a putative human disease gene built to accurately assess the influence of rare genetic changes and their phenotypic consequences in vivo.

MYCis a key oncogene overexpressed by many cancers, however, its oncogenic mechanisms are poorly understood. MYC is also central to acute lymphoblastic leukemia (ALL), the most common and second most lethal pediatric malignancy. Much of MYC's oncogenicity has been attributed to its transcription factor function, but data suggest MYC also deregulates replication in transcription-independent fashion. As a known master regulator of cancer transcriptomes and epigenomes, we hypothesize that MYC dramatically alters both gene expression and replication timing (non-random spatiotemporal process where some part of the genome replicates early, and other late) in both types of ALL - B-ALL and T-ALL. Conceivably, MYC exerts oncogenic effects upon the ALL transcription and replication programs, with some changes shared by B- and T-ALL, and others unique to only one. We aim to address two novel questions not been investigated before. First, in ALL, do the same genetic loci show aberrant RNA transcriptionandDNA replication? Second, how similar are the affected loci in two closely-related, yet distinct, ALL types driven by the same oncogene? The basis of our project is a unique double-transgenicrag2:hMYC,lck:GFPzebrafish pre-clinical model we established, which is the only animal model proven to develop both B-ALL and T-ALL. We previously showed that gene expression profiles (GEP) differentiating zebrafish B- and T-ALL also distinguish human B- and T-ALL, making this an ideal model system to study human ALL. In this model, B-ALL and T-ALL are induced by human MYC(hMYC) regulated by aD.rerio(zebrafish)rag2promoter.Since B and T lymphoblasts both expressrag2, both lineages over-express MYC, causing highly-penetrant B- and T-ALL. Differential activity of aD. rerio lckpromoter causes B cells to fluoresce dimly and T cells to fluoresce brightly, allowing us to identify and purify B-ALL and T-ALL by fluorescent microscopy and fluorescence-based flow cytometry, respectively. This unique model enables comparing B- and T-ALL in one genetic background. We have purified >20 zebrafish ALL (both T-ALL and B-ALL) and isolated their RNA and DNA. We are now analyzing RNA-seq gene expression profiles (GEP) and replication timing (RT) profiles via next generation sequencing (NGS). We will compare both ALL types to identify mRNA signatures that are unique to, or shared by, both types. We seek loci that shift DNA replication from early-to-late, or late-to-early, to define the regions that replicate at the same time in both ALL types, versus loci that vary by ALL type. We will also interrogate these data to determine whether GEP and RT profiles correlate with each other, and with known MYC target genes. In conclusion, GEP and RT have never been analyzed in the same cancer sample, or in related cancers driven by the same oncogene. Exploiting our expertise with thehMYCzebrafish model, we are delineating how MYC alters transcription and replication, to ascertain if these affect the same loci and define which loci are unique to one ALL type or shared by both. MYC hyper-activity is seen ~70% of human cancers - making MYC a crucial oncogene in human cancer biology, so our findings are likely to inform not only mechanisms operative in ALL, but also other MYC-driven cancers. Disclosures No relevant conflicts of interest to declare.

MYC is over-expressed by many cancers, yet its oncogenic mechanisms are incompletely understood. MYC is central to acute lymphoblastic leukemia (ALL) - the most common and second most lethal pediatric malignancy, and ALL afflicts even more adults. Much of MYC's oncogenic function is attributed to its role as a transcription factor, but MYC has been shown to deregulate DNA replication independent of transcription. As master regulator of transcriptomes and epigenomes, we predict that MYC impacts both biologic features in both ALL types, B- and T-ALL. We hypothesize that MYC alters both RNA expression and DNA replication (the ordered spatio-temporal process where genomic domains replicate in either early or late S-phase) in B- and T-ALL, and that these perturbations-some shared, others unique to one ALL type-drive leukemogenesis. Our project utilizes a unique double-transgenic rag2:hMYC, lck:GFP zebrafish ALL model that we established, which is the only animal model that develops both highly penetrant B- and T-ALL. In this model, B- and T-ALL are induced by human MYC (hMYC) that is regulated by a zebrafish (Danio rerio) rag2 promoter. Because B and T lymphoblasts each express rag2, both lineages over-express MYC, inducing B- and T-ALL. The differential activity of the D. rerio lck promoter (regulating GFP) causes B cells to fluoresce dimly and T cells to fluoresce brightly, permitting identification of B- vs. T-ALL by fluorescent microscopy and FACS-purification. Thus, we can compare B- and T-ALL in an isogenic background. We have collected 30 ALL samples (18 T-ALL, 12 B-ALL) and completed two types of analyses on 12 T-ALL and 3 B-ALL. Using RNA-seq, we established gene expression profiles (GEP) for both ALL types; principal component analysis and other clustering algorithms demonstrate B- and T-ALL are distinct. Although we analyze the entire transcriptome, we prioritize genes conserved in humans to focus on translatable targets. To assess DNA replication, we generated Replication Timing (RT) profiles by first FAC-sorting ALL cells based on cell cycle phase (G1, S, G2; defined by DNA content) and then performing whole-genome sequencing to generate RT profiles for the same ALL analyzed by RNA-seq. We identified differentially replicating regions by comparing RT of B-vs. T-ALL, revealing many loci where replication reproducibly shifts from early-to-late, or late-to-early, based on ALL type. Overall, despite their shared genetic driver (MYC) , we found RT differences that distinguish B- vs. T-ALL in ~30% of the genome. Most differences occur in large chromosomal domains, suggesting abnormal chromatin structure in ALL. An additional unexpected result was that many ALL G1 samples had read count differences across large chromosomal regions, indicating the presence of aneuploidies/large CNAs. Several were recurrent and lineage-specific (i.e., exclusive to B- or T-ALL). Together, our data demonstrate differences in RNA transcription, DNA replication, and regions of genomic instability that are lineage-specific, despite a shared MYC oncogene that drives both B- and T-ALL. We will next determine which deranged loci are also perturbed in human ALL, with an overarching goal of finding prognostic biomarkers and therapeutic targets. MYC hyper-activity occurs in ~70% of human malignancies. Thus, MYC is crucial to virtually all cancer biology, making our findings likely to inform not only the mechanisms that drive ALL, but also other cancers where MYC is oncogenic. Disclosures No relevant conflicts of interest to declare.