Human Reference Genome Hg19 Research Articles

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue.

AIMTo investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing.METHODSHuman iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer (OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer.RESULTSIn eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells.CONCLUSIONThe genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.

Application of whole exome sequencing in elucidating the phenotype and genotype spectrum of junctional epidermolysis bullosa: A preliminary experience of a tertiary care centre in India

BackgroundJunctional epidermolysis bullosa (JEB) is a diverse group of genodermatoses associated with extreme skin fragility. Despite several well-characterized genetic studies, molecular diagnosis of this heterogeneous group is still challenging. Recent advances in the field of genomics have seen the successful implementation of whole exome sequencing (WES) as a fast and efficient diagnostic strategy in several genodermatoses. ObjectiveIn view of the scarcity and need of molecular studies for JEB in India, we sought to explore the potential of WES in understanding the mutational spectrum of this rare, in certain subtypes lethal, sub-group of EB. MethodsWES was performed using genomic DNA from each case of EB, followed by massively parallel sequencing. Resulting reads were mapped to the human reference genome hg19. Sanger sequencing subsequently confirmed the potentially pathogenic mutations. ResultsOverall, four unrelated families (6 patients) of JEB with a highly variable clinical presentation including a rare case of LOC syndrome were studied. WES revealed 4 variations in 3 genes (LAMA3, LAMB3 and COL17A1) that are implicated in JEB. None of the variations were recurrent. In addition we proposed the probable molecular consequence of a missense mutation on the structure-function relationship of lamininβ3 protein through computational modeling studies. ConclusionsBeing the first report documenting the phenotype-genotype correlations of JEB patients from India, our preliminary experience with WES is clearly encouraging and serves as a nidus for future large-scale molecular studies to actively identify and understand JEB patients in Indian population.

Gene Expression and Alternative Splicing Datasets Analyses of MDS with Ring Sideroblasts Highlight Alternative Branchpoint Usage in Genes Involved in Iron Metabolism and Erythropoiesis

IntroductionSF3B1 hotspot mutations are associated with various cancers like uveal melanoma, chronic lymphocytic leukemia and myelodysplastic syndrome with ring sideroblasts (MDS-RS). These mutations affect RNA splicing by the use of alternative branchpoints resulting in an aberrant 3' splice site (ss) selection. RNA-sequencing (RNA-seq) analyzed to quantify exon-exon junctions identified aberrantly spliced transcripts in target genes, and half of them are predicted to be degraded by non-sense mediated decay. For this reason, target genes in SF3B1-mutated MDS remain partially characterized. In the present study, we performed deep RNA-seq analysis of bone marrow mononuclear cells in low/int-1 MDS with SF3B1 mutations to identify aberrant/cryptic splicing events among up or down-regulated gene sets.MethodsSF3B1MUT MDS (n=21) were compared to 6 SF3B1WT cases and 5 controls. Analysis of RNA-seq read count was performed using the Voom method associated with the Limma empirical Bayes analysis pipeline (https://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-2-r29). Up or downregulated gene sets were identified using Gene Set Enrichment Analysis (GSEA, false discovery rate<0.1). Gene expression profiling data (Affymetrix Hu2.0) were also available for 26/27 patient samples. TopHat (v2.0.6) was used to align the reads against the human reference genome Hg19 RefSeq (RNA sequences, GRCh37) downloaded from the UCSC Genome Browser (http://genome.ucsc.edu). Read counts for splicing junctions from junctions.bed TopHat output were considered for a differential analysis using DESeq2. Only alternative acceptor splice sites (two or more 3′ss with junctions to the same 5′ss) and alternative donor splice sites (two or more 5′ss with junctions to the same 3′ss) with P-values ≤10−5 (Benjamini-Hochberg) and absolute Log2 (fold change) ≥1 were considered.ResultsPrincipal component analysis (PCA) nicely discriminated controls from patients, and patients according to the presence of a SF3B1 mutation. A set of 6971 genes was differently expressed (P- value<0.05) between SF3B1MUT and SF3B1WT cases and allows unsupervised clustering in two separated groups (Fig. 1). Distinct gene sets also discriminated SF3B1MUT or SF3B1WT from controls.Consistent with increased amount of erythroblasts in MDS-RS bone marrows, a set of erythroid genes including several genes involved in hemebiosynthesis pathway (ALAD, UROS, ALAS2, UROD) was significantly enriched in SF3B1MUT samples. Genes selected for their involvement in the core iron-sulfur cluster mitochondrial machinery (FXN, BOLA3, FDXR, GLRX5, ISCA2, NFS1, ISCU), the iron binding and trafficking (SLC25A38, ABCB10, TFR2, SLC25A37, ABCB6, FAM132B, SLC25A39, FTH1) and the cellular iron homeostasis (ACO1, ACO2, GLRX3) were also significantly enriched (FDR<10% and nominal P-value<0.05) when input in GSEA. Moreover, other enriched gene sets were G2M checkpoint, MYC targets, oxidative phosphorylation and E2F targets. All of these observations were similarly obtained when analyzing Affymetrix data. Furthermore SF3B1MUT samples with a K700E substitution harbored a specific pattern of deregulated genes, which allowed the ordering of SF3B1MUT samples according to the type of substitution.As previously reported by AlsafadiS et al (2016), analysis of splice junctions using DESeq2 revealed an overall high level of differences between SF3B1MUT and SF3B1WTsamples. Among more than 540 differentially spliced junctions, more than 80% involved an aberrant acceptor (3'ss) site. As determined by PCA, the top 50 genes associated with relevant aberrant junctions were linked to iron metabolism or erythropoiesis and differentially expressed between SF3B1MUT and SF3B1WTsamples.ConclusionIn this study, we combined robust analyses of gene expression and aberrantly spliced transcript expression in MDS with SF3B1 mutation. By comparing SF3B1MUTversus SF3B1WT samples, we identified a set of deregulated genes in which both normally and aberrantly spliced transcripts were detected that could contribute to the physiopathology of MDS-RS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 2426: Detection of somatic mutations associated with pulmonary metastasis of osteosarcoma by whole exome sequencing

Abstract Objective: Pulmonary metastasis is a main cause of death for the patients with osteosarcoma (OS), although no specific genetic alteration associated with pulmonary metastasis has been reported. The objective of this study was to identify candidate somatic genetic alteration contributed to pulmonary metastasis in pediatric OS. Methods: Five sample sets including the tissues taken from primary tumor (P), resected pulmonary metastatic tumor (M), and normal tissue (N) of the same pediatric OS patients were analyzed by whole exome sequencing with Ion Torrent Proton sequencer. 40ng of genomic DNAs extracted from each tissue were used for multiplex PCR amplification with Ion Ampliseq Exome Kit. Data analysis including alignment to the hg19 human reference genome and variant calling was done using the Torrent Suite Software. Obtained genomic data was validated by visualizing in Integrative Genomics Viewer or capillary sequencing. Candidate somatic genetic alterations were annotated by wANNOVAR database. Pathway analysis was performed using GeneMANIA. Results: Mean read depths on the P, M, and N were 219, 212, and 109, respectively. We identified 1364 and 1311 candidate non-synonymous somatic single nucleotide variants (SNVs) among the P and M, respectively. After the validation and filtering, we detected 135 non-synonymous SNVs within the exon region in M. Among these, 32 SNVs were overlapped with those in P. Only 2 recurrent SNVs were identified within the whole SNVs. C:G&amp;gt;T:A transition mutations were frequently observed in both P and M, and the frequency were higher in M comparing to P. Pathway analysis showed a significant enrichment of the candidate SNVs in genes involved in extracellular matrix regulation (FDR;2.37E-03). Conclusion: We have identified 137 SNVs associated with pulmonary metastasis from pediatric OS. Extracellular matrix-relating signature might contribute to the genesis of pulmonary metastasis. Citation Format: Shintaro Iwata, Yasutoshi Tatsumi, Tsukasa Yonemoto, Hiroto Kamoda, Takeshi Ishii, Hiroki Nagase, Miki Ohira. Detection of somatic mutations associated with pulmonary metastasis of osteosarcoma by whole exome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2426.

Abstract 3223: Androgen receptor stimulation decreases PD-L1 expression in androgen-responsive thyroid cancer cells

Abstract Women develop thyroid cancer more often than men; however, when men develop thyroid cancer, the disease course is more lethal, with a mortality rate that is two to three fold higher than in females. Immune elimination of nascent tumor cells may explain the differences in disease occurrence/course between men and women. To address this possibility, we examined the effect of androgen on the expression of immune checkpoint molecules in an androgen responsive-thyroid cancer cell line. The undifferentiated thyroid cancer cell line, 8505C, does not express a functional androgen receptor (AR). 84E7 is a clone of 8505C that was transfected with an AR containing plasmid resulting in constitutively expressed AR. Transcriptome analysis was performed on 8505C and 84E7, with and without 5α-dihydrotestosterone (DHT) treatment. Raw sequencing reads were aligned to the UCSC hg19 human reference genome with Tophat, and Cufflinks was used to measure transcript abundances in Reads Per Kilobase of exon model per Million mapped reads (RPKM) as well as to find genes with statistically significant changes in expression as per Trapnell et al (2012). RNASeq data indicated that DHT treatment of 8505C cells does not result in a significant change in gene expression (only 14 of 23,285 genes), consistent with the lack of a functional androgen receptor. In contrast, DHT treatment of 84E7 resulted in &amp;gt;2 fold expression changes in 1,552 genes. Examination of the immune checkpoint molecules CD28, CD80, CD86, CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, TIM-3L, 4-1BB, 4-1BBL, OX40, and OX40L revealed that in DHT-treated 84E7 cells, PD-L1 was the sole immune checkpoint molecule that exhibited a significant expression change with a 1.8 log2 fold decrease (p = 0, q = 0), or 72% reduction in mRNA content. This is potentially significant as PD-L1 is produced by tumor cells as a strategy for subverting and evading the immune response, specifically T cells, allowing for continued tumor growth and metastases. In the thyroid, the presence of androgen-activated androgen receptors could perhaps lead to an environment that is more favorable for the immune system and may help eliminate nascent thyroid cancer cells. Thus, men may experience a decreased incidence of thyroid cancer due to an enhanced (less inhibited) anti-tumor environment. However, failure of this system and a subsequent increase in PD-L1 expression may help to explain the onset and severity of thyroid cancer in males. This finding opens the possibility for the use of immune checkpoint inhibitors in the treatment of thyroid cancer. Citation Format: Timmy O’Connell, Melanie Jones, Anvita Gupta, Augustine Moscatello, Raj K. Tiwari, Jan Geliebter. Androgen receptor stimulation decreases PD-L1 expression in androgen-responsive thyroid cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3223.

Abstract 78: Targeted next-generation resequencing in pediatric osteosarcoma patients

Abstract Background: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Recent genomic analysis demonstrated that OS are characterized as a high frequency of structural variations and chromosomal copy number changes. Meanwhile, specific genomic alterations such as RB1 or TP53 have been identified, although driver gene mutation for OS tumorigenesis has yet to be found. The aim of this study is to identify specific somatic mutations in OS patients by the method of comprehensive targeted resequencing. Patients and Methods: 13 pairs of tumor and non-tumor frozen tissues taken from the patients diagnosed as a primary conventional OS were subjected to this study. All tumor samples were collected at the time of preoperative biopsy and confirmed pathologically the tumor cell content more than 80%. We utilized a next generation sequencing technique with the Ion AmpliSeq Comprehensive Cancer Panel (CCP) containing 409 cancer-related genes. 40 ng of DNAs were used for multiplex PCR amplification and sequencing was run on the Ion Torrent Proton loaded with PIv2 chip. Data analysis including alignment to the hg19 human reference genome and variant calling was done using the Torrent Suite Software. Obtained genomic data was validated by visualizing in Integrative Genomics Viewer and candidate mutations were annotated by wANNOVAR database. Results: An adequate library was obtained from all samples for subsequent sequencing. Mean read length was 110 base pairs and an average coverage was approx.1500 for tumors and 400 for paired non-tumors. A total of 153 candidate non-synonymous somatic SNVs (median; 13.9 per tumor) were found with the threshold of over 5% frequency. After validation analysis and filtering process of variants, at least 12 SNVs were confirmed in 8 patients. Recurrent SNVs have not been observed among the 13 patients. There was a trend toward increased number of SNVs in dead patients comparing to alive patients (median 0 vs 1), although no significant difference was observed (P = .23, Wilcoxon test). Pathway analysis of the candidate SNVs showed significant correlations with three gene function categories including cell cycle, ATP binding and ligand-dependent nuclear receptors (p&amp;lt;0.02). We also calculated relative copy number changes in tumors using the coverage analysis data and found at least three gene amplification on chromosome 1p, 17p and 19p. Conclusion: We identified at least 12 non-synonymous somatic SNVs of cancer-related genes on the CCP panel in pediatric OS patients. Targeted sequencing is a useful and cost-effective tool to quickly survey known cancer-related mutations of pediatric OS. Citation Format: Shintaro Iwata, Yasutoshi Tatsumi, Tsukasa Yonemoto, Hiroki Nagase, Hiroto Kamoda, Takeshi Ishii, Miki Ohira. Targeted next-generation resequencing in pediatric osteosarcoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 78. doi:10.1158/1538-7445.AM2015-78

Abstract 4137: Clonal evolution defines the natural history of metastatic pancreatic cancer

Abstract INTRO: Pancreatic adenocarcinoma (PDAC) patients are often diagnosed with metastatic disease, yet the extent to which metastasis is determined by genetic evolution is unknown. A better understanding of this lethal process would have great clinical significance. METHODS: We selected four patients from the Johns Hopkins Rapid Medical Donation Program using strict criteria that included diagnosis with Stage IV disease and no treatment prior to death. Tissues from each patient were macrodissected to reduce stromal cell contamination and genomic DNA was extracted and quantified. A set of high quality primary or metastatic samples from these four patients (n = 35, range 4-11 per patient) were whole genome sequenced using the Illumina Hi-Seq 2000 platform, ultimately generating a raw list of 165,815 potential variants. After alignment to the hg19 human reference genome, the variants were filtered for single nucleotide polymorphisms, sequence directionality, and allele frequency in order to limit sequencing artifacts while maximizing detection of clonal changes. Subsequently, we selected 3256 somatic variants for targeted resequencing of the original 35 samples and in an additional 146 samples of the primary carcinoma or metastases (range 5-53 additional samples per patient). RESULTS: Whole genome sequencing resulted in a median high quality distinct coverage of 63X per genome and a median effective coverage of 97.9%. Point mutations in KRAS were identified at allele frequencies ranging from 8-88%. Targeted sequencing resulted in a median distinct coverage depth of 189X across all samples. We developed a robust statistical method to determine the mutation status across the samples of a patient based on an estimated false positive rate of the targeted sequencing of 0.23%. After discarding samples with low purity or low coverage, we found high homogeneity in the mutation profiles across samples from the primary tumor and samples from different metastases in each patient. We show that for many metastases there exists a corresponding sample from the primary tumor differing only in a few positions. The majority of mutations were either present in all or in only one sample of a patient. The low number of remaining parsimony-informative mutations prevents the generation of precise evolutionary trees. However, using hierarchical clustering and our newly developed phylogenetic method, we were able to infer basic evolutionary relationships among the samples. CONCLUSIONS: The natural history of metastatic PDAC is defined by the accumulation of a large number of mutations within the primary tumor, including most of the driver mutations. Indeed, evolutionary analyses indicate that metastases have relatively few unique mutations and are by-products of clonal evolution within the primary tumor over a prolonged period. Citation Format: Alvin P. Makohon-Moore, Ming Zhang, Johannes G. Reiter, Ivana Bozic, Fay Wong, Yuchen Jiao, Krishnendu Chatterjee, Martin Nowak, Nickolas Papadopoulos, Bert Vogelstein, Kenneth W. Kinzler, Christine A. Iacobuzio-Donahue. Clonal evolution defines the natural history of metastatic pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4137. doi:10.1158/1538-7445.AM2015-4137

Identification of Target Genes Regulated by KSHV miRNAs in KSHV-Infected Lymphoma Cells.

This study aimed to identify target genes regulated by KSHV miRNAs in KSHV-infected lymphoma cells. Original Ago HITS-CLIP data of BC-3 and BCBL-1 cell lines were downloaded from SRA database in NCBI, including mRNA and miRNA samples. The raw mRNA reads were mapped into human reference genome hg19 via TopHat for read alignment. PCR duplicates were removed via the SAM tool and the peaks of reads were analyzed via Cufflinks. For miRNA data, the raw data were mapped to the mature miRNA sequences based on miRBase via Bowtie. Peak intersection was computed by using intersectBed in BEDtools. Then, the mature miRNA seeds were identified and then were aligned with 3' UTR merged peaks. The regulationships of miRNAs and their corresponding genes were analyzed based on the signal of RNA-induced silencing complex. Totally, 7 KSHV-related genes regulated by KSHV miRNAs were identified, including IPO5, EDA, NT5C3, WSB1, KCNS1, PRAM1 and MTRNR2L6. Among them, EDA, MTRNR2L6 and IPO5 were regulated by multiple KSHV miRNAs, such as kshv-miR-K12-1-5p, kshv-miR-K12-4-3p and kshv-miR-K12-3-5p, respectively. Furthermore, expression of kshv-miR-K12-1-5p and kshv-miR-K12-3-5p in BCBL-1 cell line were lower than that in BC-3 cell line, conversely, expression of kshv-miR-K12-4-3p in BCBL-1 cell line were higher than that in BC-3 cell line. In conclusion, potential target genes regulated by KSHV miRNAs in KSHV-infected lymphoma cells might play key roles in the nosogenesis of this disease. These findings might provide the basis for deep understanding of KSHV-infected tumors and further molecular experiments.

The Use of RNA Sequencing to Identify Disease-Specific Gene Expression Signatures and Critical Regulatory Networks Across Hematologic Malignancies

IntroductionTranscriptome analysis by next-generation sequencing (RNA-seq) allows investigation of hematologic malignancies at unsurpassed resolution and provides promising insights into their molecular etiology. Dissecting the underlying biology depends on specific molecular signatures deregulated in the disease subtypes. In addition, gene expression profiles may potentially be used to identify driver genetic alterations and stratify patients based on molecular subtype. In this study, we generated gene expression profiles from RNA-seq data derived from patients with hematologic disease, including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and multiple myeloma (MM). Using these data, we aimed to identify disease-specific differentially expressed genes and gain better understanding of the biological functions of these genes for the development of biomarkers and therapeutic strategies in these malignancies.MethodsMononuclear cells (MNCs) were isolated from bone marrow or peripheral blood by Ficoll separation from AML (n=24), ALL (n=5), CMML (n=10), MDS (n=4), CML (n=2) and MM (n=7) patients. For MM, CD138+ cells were enriched from MNCs by immunomagnetic bead selection. Total RNA from isolated cells was depleted of ribosomal RNA and reverse transcribed for cDNA. RNA-seq libraries were prepared and sequenced on the Illumina HiSeq instrument. Reads were filtered and aligned to the hg19 human reference genome using TopHat. Second step normalization was carried out to compare gene expression values (FPKM) across samples using a normalization factor derived from 18 reference genes. A log normalized relative expression value for each gene was calculated compared to the median value across all samples. Average linkage based hierarchical clustering was performed and then visualized with TreeView. Network and pathway analyses were performed with IPA (www.ingenuity.com) and Cytoscape¨.ResultsUnsupervised hierarchical clustering of all samples resulted in grouping based on clinical phenotypes with a unique gene signature characteristic for each group (Figure). Analyses of different hematologic malignancies ensured credibility of the classification and highlighted differences in underlying cell signaling networks of each disease. Group I consisted of the multiple myeloma samples, where we identified 25 frequently upregulated genes. Network analyses revealed expression of the upregulated molecules is controlled by two major transcription factors, IRF4 and JUN, which represent the major hubs of the gene signature network. Group II consisted of samples with the BCR-ABL1 fusion and BCR-ABL-like ALLs. Enriched genes in this subgroup included regulators of B-cell development and maturation, plus genes involved in antigen presentation including TCL1A, CD19, CD79, HLA-DQA1, HLA-DQB1 and HLA-DRB1. Group III represented myelodysplastic and myeloproliferative neoplasms and included the MDS and CMML samples. A set of 14 genes differentially enriched in this group formed a unique pro-inflammatory signature. This included TNF-α and IL1B, which act as major regulators of smoldering inflammation driving NF-κB activity and orchestrating downstream activation of signature genes. While MM is known to have activated NF-κB signaling, the gene expression signatures of the MM and MDS/MPN groups were distinct from each other, and included activation of separate sets of cytokines and chemokines. AML samples exhibited heterogeneity in gene expression and formed two groups (IVA, IVB). A HOX gene family expression signature was observed in the FLT3-ITD positive AML samples.SummaryOur results show that RNA-seq can be used to identify dominant gene expression patterns characterizing different hematologic disease samples, including those sharing a common genetic base (e.g. BCR-ABL, FLT3-ITD) or clinical phenotype (e.g. MDS/MPN, MM). Based on our results, IRF4 may be an attractive therapeutic target for MM. CMML is difficult to diagnose, however, it can be defined by a set of differentially expressed genes that could potentially be used as diagnostic markers. We also show a pivotal role for NF-κB and TNF-α signaling in the pathogenesis of MDS/MPN suggesting that drugs targeting these factors may be useful for the treatment of these diseases. [Display omitted] DisclosuresPorkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Heckman:Celgene: Research Funding.

A de novo germline mutation of APC for inheritable colon cancer in a Chinese family using multigene next generation sequencing

Inheritable colorectal cancers (CRC) accounted for about 20% of the CRC cases, such as hereditary nonpolyposis colorectal cancer (HNPCC), Gardner syndrome and familial adenomatous polyposis (FAP). A four-generation Han Chinese family was found affected with polyposis in colons. Inferred from the pedigree structure, the disease in this family showed an autosomal dominant inheritance model. To locate the causal mutations in this family, genomic DNAs were extracted and the next generation sequencing for 5 genes relating to colon cancer performed by Ion Torrent Personal Genome Machine with a 314 chip. The reads were aligned with human reference genome hg19 to call variants in the 5 genes. After analysis, 14 variants were detected in the sequenced sample and 13 been collected in dbSNP database and assigned with a rs identification number. In these variants, 9 were synonymous, 4 missense and 1 non-sense. In them, 2 rare variants (c.694C>T in APC and c.1690A>G in MSH2) might be the putative causal mutations for familial adenomatous polyposis (FAP) since the rarity of the mutated allele in normal controls. c.694C>T was detected in only affected members and generated a premature stop codon in APC. It should be a de novo germline mutation making APC containing this stop codon as targets for nonsense-mediated mRNA decay (NMD). c.1690A>G in MSH2 was not only detected in affected members, but also in normal ones in the family. Functional prediction revealed that the amino acid affected by this variant had no effect on the function of MSH2. Here, we report a de novo germline mutation of APC as the causal variant in a Chinese family with inheritable colon cancer by the next generation sequencing.

Somatic Structural Variations in Pediatric High Hyperdiploid Acute Lymphoblastic Leukemia Revealed by Paired-End Parallel Sequencing

Abstract 401 Introduction:High hyperdiploidy (51–67 chromosomes) is the most frequent numerical cytogenetic alteration found in pediatric B-cell precursor acute lymphoblastic leukemia (ALL), occurring in 25–30% of patients. It is characterized by nonrandom gains of chromosomes X, 4, 6, 10, 14, 17, 18, or 21. Children suffering from high hyperdiploid ALL have a good prognosis, nevertheless in 15–20% of cases the disease will recur. The mechanisms involved in the pathogenesis of primary and relapsing high hyperdiploid ALL are poorly understood. In some cases, IGH rearrangements arise in utero, indicating an early formation of pre-leukemic clones. However, the cellular origin of these pre-leukemic clones, as well as the molecular mechanism underlying the formation of high hyperdiploid cells, remains to be determined. Further genetic changes assisting in the development of ALL and recurrent disease are still unknown. Objective:By using massive parallel genome-wide next generation sequencing (Illumina/Solexa), we intended to identify specific cytogenetic structural variations (SVs) of high hyperdiploid ALL and possible clonal relationships between paired diagnostic and relapse ALL samples. Method:Paired-end sequencing libraries were generated from genomic DNA of diagnostic and relapse leukemic samples as well as germline DNA from the same patient. Libraries of two patients and one high hyperdiploid ALL cell line (MHH-CALL-2) with insert sizes of 350–400 bp were sequenced with paired end reads. Read lengths of 36 bp (Genome analyzer IIx) or 51 bp (HiSeq 2000) were sequenced, respectively. Sequencing raw data were aligned to the human reference genome hg19 (GRCh 37) by Burrows-Wheeler Aligner (BWA) and duplicate reads were removed. Copy number variants (CNVs), deletions, intrachromosomal inversions and interchromosomal translocations were analyzed by FREEC and GASV. After subtraction of germline SVs, putative leukemia-specific SVs were obtained. These were validated by PCR performed on genomic DNA. Specific breakpoints of SVs at single base resolution were identified by capillary sequencing of the PCR products. Results:Sequencing of different libraries yielded 95–279 million unique reads that mapped with both ends to the reference genome. Sequence coverages of 57–87% and fragment coverages of 4.9–12.3x were achieved (Table 1). [Display omitted] CNV profiles with 10 kb resolution were generated. A comparison of the CNVs of diagnosis and relapse ALL samples demonstrated a high degree of conformity with only few additional alterations present mainly, but not exclusively, in the relapse samples. In one of the patients, a large gain of chromosome 1q was only observed in the relapse sample (Figure 1). [Display omitted] SV analysis of all samples resulted in a total of 375 intragenic deletions, 16 intergenic inversions and 83 translocations (Table 1). PCR validation identified 2 previously unknown somatic translocations in the MHH-CALL-2 cell line concerning chromosomes 3 and 7 as well as chromosomes 15 and 18. Furthermore, 6 novel translocations present at diagnosis and relapse could be validated in patient samples. They were concerning chromosomes 3, 11, 12 and 20. One unique new relapse-specific translocation t(4;7) was identified. Conclusion:Paired-end sequencing of leukemia samples and matched non-tumor materials provides a robust tool for the discovery of genome-wide structural rearrangements. The high degree of conformity of CNVs and SVs detected in paired diagnosis/relapse samples indicate a common origin and a close relationship of the leukemic clones at diagnosis and relapse. The observation of few additional alterations in both diagnostic and relapse samples suggests the presence of different subclones at the time of diagnosis and the evolution of the relapse clone from either the diagnostic clone or a minor subclone. Disclosures:No relevant conflicts of interest to declare.