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Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using “next-generation” sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chroni myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. “Deeper” sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34+ and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Purpose Interstitial deletions of chromosome 5q are common in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), pointing toward the pathogenic role of this region in disease phenotype and clonal evolution. The higher level of resolution of single-nucleotide polymorphism array (SNP-A) karyotyping may be used to find cryptic abnormalities and to precisely define the topographic features of the genomic lesions, allowing for more accurate clinical correlations. Patients and Methods We analyzed high-density SNP-A karyotyping at diagnosis for a cohort of 1,155 clinically well-annotated patients with malignant myeloid disorders. Results We identified chromosome 5q deletions in 142 (12%) of 1,155 patients and uniparental disomy segments (UPD) in four (0.35%) of 1,155 patients. Patients with deletions involving the centromeric and telomeric extremes of 5q have a more aggressive disease phenotype and additional chromosomal lesions. Lesions not involving the centromeric or telomeric extremes of 5q are not exclusive to 5q− syndrome but can be associated with other less aggressive forms of MDS. In addition, larger 5q deletions are associated with either del(17p) or UPD17p. In 31 of 33 patients with del(5q) AML, either a deletion involving the centromeric and/or telomeric regions or heterozygous mutations in NPM1 or MAML1 located in 5q35 were present. Conclusion Our results suggest that the extent of the affected region on 5q determines clinical characteristics that can be further modified by heterozygous mutations present in the telomeric extreme.

Datasets with a high dimensional feature space, advancing statistical methods, and computational efficiency were analyzed to uncover the rules of the circadian rhythms. The aim of the study was to uncover the identity, the dynamic behavior, and the interactions among the components of the circadian clock. Transcriptional profiling has exposed the regulon conferring benefits for circadian biology and bioinformatics. Circadian plant time course gene expression data was examined, this was the prerequisite for Naive Bayes classifiers which were trained and led to expression model with a success rate of up to 87%. The model showed new combinatorial rules, including presence of elements and their frequencies in driving particular phases. Implementation of Genemining V2.3 multipotent algorithm showed the specific combinations of elements responsible for expression patterns, highlighting the role of GATA motifs. State-of-the-art technologies allowed for a model in silico, the first such model was made using time course circadian data.

Datasets with a high dimensional feature space, advancing statistical methods, and computational efficiency were analyzed to uncover the rules of the circadian rhythms. The aim of the study was to uncover the identity, the dynamic behavior, and the interactions among the components of the circadian clock. Transcriptional profiling has exposed the regulon conferring benefits for circadian biology and bioinformatics. Circadian plant time course gene expression data was examined, this was the prerequisite for Naive Bayes classifiers which were trained and led to expression model with a success rate of up to 87%. The model showed new combinatorial rules, including presence of elements and their frequencies in driving particular phases. Implementation of Genemining V2.3 multipotent algorithm showed the specific combinations of elements responsible for expression patterns, highlighting the role of GATA motifs. State-of-the-art technologies allowed for a model in silico, the first such model was made using time course circadian data.

While deletion of the long arm of chromosome 7 (del(7q)) along with monosomy 7 (-7) are common in myeloid neoplasms and especially MDS, their associated pathogenetic consequences and the genes responsible for the clinico-morphologic phenotypes remain unknown. To characterize the molecular defects resultant from del(7q), we applied a combined analysis of SNP-array karyotyping, whole exome NGS, targeted deep NGS and deep whole RNA NGS to facilitate identification of somatic mutations, loss of heterozygosity (LOH) and haploinsufficiency. First, using a cohort of 1595 patients, we precisely defined 3 commonly deleted regions in 7q22, 7q34, and 7q35-36. To identify genes involved in the pathogenesis of del(7q), we applied whole exome NGS to 428 patients with MDS and related conditions, including 72 cases of -7/del(7q) or UPD7. We found that both recurrent deletions with haploinsufficient expression and somatic nonsense/frameshift mutations were present in the genes CUX1 (7q22), LUC7L2 (7q34) and EZH2 (7q36), located on 7q. For this project we focused our study on the LUC7L2 gene encoding a spliceosomal protein that interacts with U1 snRNP to recognize 5’ splice sites. This function is in contrast to other spliceosomal genes mutated in MDS, such as SF3B1, U2AF1, SRSF2 and SF3A1that interact with the U2 snRNP complex and the 3’ splice site. Initially, we found 8 cases of LUC7L2 mutation including 2 cases of hemizygous and 1 case of homozygous mutations; all LUC7L2 mutations result in premature stop codons. The concomitant mutational spectrum in LUC7L2 mutant cases differed from those with EZH2 or CUX1 mutations. For example, TP53 and TET2 are mostly mutually exclusive with LUC7L2 mutations. While LUC7L2 mutant cases were low grade-MDS (RCUD or RCMD) or CMML, the survival impact of LUC7L2 mutation was similar to that seen in -7/del(7q) (LUC7L2; HR=2.36). Most of the mutations are heterozygous (diploid chr7) or homozygous (UPD7q). However, in addition to a few hemizygous mutations, wild type expression of LUC7L2 is haploinsufficient in del(7q). In total we found 117 cases (9%) with del(7q) and resultant decreased expression of LUC7L2, showing comparably poor survival as in the mutant cases (HR=1.99). Next, we hypothesized that dysfunction of LUC7L2 causes splicing defects (missplicing) in specific genes involved in leukemogenesis. Using deep RNA NGS we compared splicing patterns of 201,837 exons between the cases with deficient function of LUC7L2 (mutation/low-expression; n=11) versus wild-type (n=11), and identified concordant alternative splicing patterns in 44 genes (increased exon skipping in 27 genes; increased exon retention in 17 genes). For example, we found abnormal splicing of genes involved in functionally important pathways including the RAS pathway (NF1) and the TGF-β pathway (SMAD5). In cases of mutation/low-expression of LUC7L2, as a result of increased skipping of NF1 exon 31(NM_001042492), the type I isoform of NF1 predominates as compared to wild type (types I/II ratio; 0.76 vs. 0.45, p In conclusion, novel somatic mutations of LUC7L2 suggest that it could be a candidate gene associated with the poor prognosis of -7/del(7q) and UPD7. Loss of function or low expression of LUC7L2 results in distinctly altered splicing patterns involving genes associated with proliferation or leukemogenesis. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

Abstract 121 Balanced and unbalanced chromosomal lesions and genetic mutations are hallmarks of myeloid malignancies. In addition, aberrant methylation of CpG islands, leading to epigenetic silencing appears to play a significant role in tumor suppressor gene inactivation and malignant progression. While various mechanisms of chromosomal instability have been identified, the pathogenesis of epigenetic instability remains unexplored. ASXL1, EZH2 and TET2 mutations, found in myeloid disorders provide a potential link between genetic and epigenetic events. UTX is a histone H3K27 demethylase belonging to the polycomb group of proteins. Methylation at lysine 27 correlates with gene silencing and repression. A recent mutational screen of cancer cell lines has identified mutations in UTX in AML THP-1 cells. We screened a series of myeloid disorders for UTX gene mutations and identified an index case in a CMML patient with a somatic UTX missense mutation. This sample demonstrated × chromosome copy neutral loss of heterozygosity (CN-LOH) that included UTX on a whole genome SNP-A array. UTX was then sequenced in 49 additional patients with MDS/MPN and 24 with secondary AML. Two of the MDS/MPN (2/49, 4%) and 3 of the sAML patients (3/24; 12%) showed UTX mutation. Three previously unreported missense polymorphisms were noted (present in paired CD3+ cells and detected with a frequency of UTX mutation and dysfunction might be predicted to lead to altered histone repression marks and alterations in epigenetic regulation. Consistent with this hypothesis, we tested for H3K27-Me3 methylation by ELISA and observed a 45% methylation increase in a UTX mutant patient sample. To further investigate the function of UTX, consequences and treatment implications of the corresponding mutations we transduced THP-1 cells with a lentiviral vector containing the UTX cDNA (UTX+) or an empty vector. Following clone selection, the resultant UTX+ cell line showed 1000-fold UTX mRNA increase and distinct protein overexpression by western blot (WB), as compared to the baseline UTX null cells. Proliferation kinetics up to 120h indicated that overexpression of UTX results in increased cell proliferation by 20% compared to controls. Treatment with decitabine (120h; 0.5μ M) resulted in a differential effect on UTX+ cells: proliferation increased 4.6-fold vs. 1.3-fold in UTX null cells. Based on the hypothesis that mutation in UTX may lead to epigenetic instability and accumulation of aberrant methylated CpG sites, we further investigated epigenetic changes in UTX+ and UTX null cells. WB showed comparable levels of global H3K27-Me3 and H3 acetylation levels in UTX+ and UTX null cells, and only modest decreases (9%) of trimethylation in a more precise ELISA assay. Similarly, when methylation array (14K genes; 27K CpG sites) was used to assess the effects of UTX overexpression, global methylation levels expressed by averaged β-values did not significantly differ. However, 153 specific CpG sites were found to be differentially methylated on the array: with 68 genes hypo- and 85 hypermethylated. In particular SSRP1, NCOR2 and DIRAS3 genes showed hypomethylated promoters in the UTX+ cells, suggesting that UTX is involved in gene specific demethylation. In sum, our results suggest that UTX gene may be involved in epigenetic regulation of promoters through site-specific histone demethylation function. UTX mutation may compromise this function, thereby promoting repression of tumor suppressor genes. Disclosures: No relevant conflicts of interest to declare.