Добірка наукової літератури з теми "Exon-spanning reads"

e23521 Background: Ewing family tumors including Ewing sarcoma (ES) and desmoplastic small round cell tumor (DSRCT) are characterized by EWSR1 and ETS fusion partners including EWS-FLI1, EWS-ERG, EWS-WT1, and others. Since 2019 our Next Generation Sequencing (NGS) sarcoma panel (N = 338) identified both fusion partners and exons containing the EWS breakpoint in ES and DSRCT. Hence, it should be possible to learn more about the exact breakpoints of EWS fusion genes. The molecular diversity and functionality of these fusion transcripts, especially the exact sequence, which could be identical, similar, or unique, may have significant biological implications for diagnostics and treatment options. We have used in-frame analysis of EWS gene fusion breakpoints to identify corresponding polypeptides spanning the breakpoints of the most common EWS gene fusions to provide useful insights about ES and DSRCT. Methods: Pathology reports and EWS gene fusions were analyzed using the Cleveland Clinic NGS panel which is based on anchored multiplex polymerase chain reaction (PCR) enriched for 34 gene targets. The amplicons were subjected to massively parallel sequencing with 151x2 cycle pair-end reads. An informatics pipeline was used for read alignment (GRCh37 as reference genome), fusion identification, and annotation. The DNA sequence across fusion junction and translational amino acid sequences were extracted for comparison in a preliminary teaching set (37/338 EWS fusions). Results: EWS-FLI1 fusions at exon 7-7 (chr22:29683123-chr11:128675261) and exon 7-6 (chr22:29683123-chr11:128651853) were the most common fusion genes in ES (N = 24/32;75%). Other ES gene fusions included exons 10-6, 7-10, 9-8, 7-2 EWS-FEV, 7-8 EWS-ERG, 7-9 EWS-ERG, and 10-9 EWS-ERG. EWS-WT1 gene fusions (chr22:29683123-chr11:32414301) in DSRCT exon 7-7 occurred in 4 of 5 cases (80%). In frame analysis of the common gene fusions in 75% of ES and DSRCT (total n = 37) yielded identical corresponding polypeptides that span the breakpoint (table). Conclusions: Analysis of ES and DSRCT fusion genes provides evidence that cancer-specific fusion genes are associated with common identical breakpoints and corresponding in-frame polypeptides. Our results show that the identification of in-frame polypeptides from the fusion gene sequencing can identify potential nested sets of cancer-specific mRNAs and polypeptides. This information may become relevant for diagnostic and therapeutic targets future vaccines and diagnostics against ES and DSRCT as well as other cancers characterized by fusion genes or frameshift mutations. [Table: see text]

Abstract Background: IMiDs cytotoxicity in MM cells is mediated through their binding to CRBN within the cullin ring (CRL4) ligase. This binding triggers the ubiquitylation and proteasomal degradation of IKZF1/3. CRBN thalidmomide binding domain (TBD) was mapped to its C-terminus and the crystal structure of the CRBN-IMiDs bound complex identified several aa within exons 10 and 11 as essential for the IMiDs glutarimide ring binding to CRBN. Several groups including ours have reported that loss of CRBN is associated with resistance to IMiDs, however this does not appear to be the sole mechanism of resistance in primary MM cells. Furthermore, and while CRBN mutants (Y384A and W386A) are defective for IMiDs binding, acquisition of CRBN mutation is a rare event in MM patients suggesting alternative mechanisms of resistance. Thirteen splice variants of CRBN are reported (ensembl.org), however to date it is unclear whether these isoforms are expressed as proteins and their contribution to IMiDs resistance is yet to be defined. Methods and Results: In this study we investigated whether expression of full length CRBN (FL-CRBN) relative to its variants lacking the TBD and particularly the splice variant CRBN-005 (ENST00000424814) lacking exon 10, contribute to IMiDs resistance. RNA-seq analysis was performed on CD138 sorted cells in 15 paired patients samples obtained sequentially prior to lenalidomide treatment initiation and after development of resistance. Transcriptome sequence data was generated by RNA-seq with a minimum of 70x106 reads per sample. Filtered Fastq files were processed with the splice aligner TopHat against hg19. Of interest, splice isoforms of CRBN including isoforms lacking exon 10 and to a lesser extent exon 8 were identified in nearly all patients, albeit with at variable frequency. Splicing almost universally involved the full length of exon 10 including aa W382 and H378 that are now recognized to bind the 2 carbonyls residues on the IMiDs glutarimide ring and hence required for IMiDs binding to CRBN. Of note, mutation analysis of these 30 samples using the GATK RNAseq pipeline did not identify any mutations within CRBN exons 10 or 11. Furthermore, exome sequencing of CD138 cells from 10 additional lenalidomide resistant patients did not identify any CRBN SNVs or indels confirming the rarity of this event. In order to assess the contribution of FL-CRBN transcript and/or its splice variant (CRBN-005) to IMiDs sensitivity, we first confirmed by qRT-PCR (n=26, amplicons with 2 sets of primers overlapping exons 8-9 and exons 10-11) that low pre-treatment CRBN levels was significantly associated with shorter PFS (p=0.008) to lenalidomide. We next compared FL-CRBN (probe spanning exons 10-11) and CRBN-005 (Taqman probe spanning exons 9-11 junction) mRNA expression (qRT-PCR) in paired samples (n=21 patients - 42 pairs) collected immediately pre-treatment and at the time of progression post-lenalidomide. In 9/21 (42.8%), a significant reduction (2-ΔΔCT < 0.75) in the FL-CRBN amplicon levels was observed between the paired pre- and post-treatment samples. The ratio of spliced CRBN-005 to full length CRBN (CRBN-005 / FL-CRBN) was significantly higher (1.3 to 54 fold) at the time of relapse in 11/21 (52.3%), including 5 patients where CRBN-FL transcript levels were unchanged. Lastly, to confirm whether CRBN-005 expresses a stable protein and to evaluate its role in IMiDs resistance, we cloned spliced CRBN-005 isoform (Δ10-CRBN) or full length CRBN (WT-CRBN) into pcDNA3 plasmid and transfected them in HEK293T cells. The Δ10-CRBN and WT-CRBN plasmids expressed a ~ 45 and 51 kDa proteins respectively that were detectable by western blotting with CRBN65 antibody (Celgene). Functionally, we co-transfected HEK293T cells with a lentiviral plasmid expressing Aiolos and the Δ10-CRBN or WT-CRBN plasmids. While treatment of WT-CRBN expressing cells with lenalidomide resulted in full loss of Aiolos, the expression of Δ10-CRBN significantly mitigated this effect. Conclusions: Study of the transcriptome of paired pre- and post-IMIDs in myeloma primary cells confirms the expression of CRBN-005 splice isoform lacking the IMiDs binding domain and reveals its enrichment in a subset of IMiDs resistance patients. Functionally we have demonstrated a novel mechanism of IMiDs resistance where the spliced isoform CRBN-005 acts as a dominant negative blocking IMiDs binding the CRL4 E3 ligase. Disclosures Bahlis: Celgene: Honoraria, Research Funding.

Abstract Abstract 884 Blast crisis is the terminal phase of chronic myeloid leukemia (CML) with a short median survival of approximately six months. At present, little is known about molecular mechanisms underlying disease progression. We hypothesized that mutations occurring in other myeloid and lymphatic malignancies are acquired during disease progression from chronic phase to blast crisis. Here, in total 40 blast crisis CML cases (n=25 myeloid, n=10 lymphoid, n=5 not specified) were analyzed, all diagnosed between 9/2005 and 7/2009. First, all cases were investigated for IKZF1 deletions by PCR using specific primer pairs for the common intragenic deletions spanning from exon 2–7, or exon 4–7 as published by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, in 17.5% (7/40) of cases intragenic IKZF1 deletions were detected. Secondly, next-generation deep-sequencing (454 Life Sciences, Branford, CT) was used to investigate 11 candidate genes in all 40 patients for a broad molecular screening. Known hotspot regions were sequenced for CBL (exons 8 and 9), NRAS (exons 2 and 3), KRAS (exons 2 and 3), IDH1 (exon 4), IDH2 (exon 4), and NPM1 (exon 12). Complete coding regions were analyzed for RUNX1, TET2, WT1, and TP53. To perform this comprehensive study, amplicon-based deep-sequencing was applied using the small volume Titanium chemistry assay. To cope with the great number of amplicons, in total 59, 48.48 Access Arrays were applied (Fluidigm, South San Francisco, CA), amplifying and barcode-tagging 48 amplicons across 48 samples in one single array (2,304 reactions). In median, 430 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. Further, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. In summary, after excluding known polymorphisms and silent mutations in 33/40 patients 53 mutations were identified: RUNX1 (16/40; 40.0%), ASXL1 (12/40; 30.0%), WT1 (6/40; 15.0%), NRAS (2/40; 5.0%), KRAS (2/40; 5.0%), TET2 (3/40; 7.5%), CBL (1/40; 2.5%), TP53 (1/40; 2.5%), IDH1 (3/40; 7.5%), IDH2 (0/40), and NPM1 (0/40). Thus, 82.5% of blast crisis CML patients harbored at least one molecular aberration. In median, one affected gene per patient was observed (range 1–5). In detail, RUNX1 was associated with additional mutations in other genes, i.e. 9/16 cases were harboring additional mutations in combination with RUNX1. Similarly, in 8/12 patients with ASXL1 mutations additional aberrations were detected. With respect to myeloid or lymphoid features ASXL1 mutations (n=11) were exclusively observed in patients with myeloid blast crisis (n=1 not specified), in contrast 5/7 IKZF1 cases were detected in cases with lymphoid features (n=1 myeloid, n=1 not specified). Interestingly, besides IKZF1 (n=5) and RUNX1 (n=3) alterations there was no other mutated gene occurring in lymphoid blast crisis CML. In addition, no aberration was detected in NPM1, and in contrast to published data, in our cohort only one patient harbored a mutation in TP53. Moreover, for 8 patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1 (n=1), WT1 (n=2), and IDH1 (n=2), matched DNA from the initial diagnosis at chronic state was available. In these specimens respective IKZF1 deletions, RUNX1, and ASXL1 mutations were not detectable indicating that IKZF1, RUNX1, and ASXL1 mutations had been developed during disease progression and act as driver mutations in these cases. WT1 and IDH1 mutations occurred at first diagnosis in one case each, indicating these genes would constitute passenger mutations. In conclusion, this comprehensive study on 12 molecular markers enabled to characterize for the first time that 82.5% of blast crisis CML cases harbor specific molecular mutations. IKZF1 and RUNX1 alterations were identified as important markers of disease progression from chronic state to blast crisis. Moreover, technically, a novel combination of a high-throughput sample preparation assay for targeted PCR-based next-generation deep-sequencing was developed and allowed to broaden our molecular understanding in blast crisis CML. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Wille:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.

Abstract Abstract 1035 Mutations of the ten eleven translocation (TET2) gene have been reported to be frequent in hematological malignancies. However, data are preliminary and investigation is challenging and labor-intensive with standard sequencing techniques. Here, we used massively parallel Titanium amplicon next-generation sequencing (NGS) technology (454 Life Sciences, Branford, CT) and investigated 76 patients with acute myeloid leukemia (AML), including 66 de novo AML, 6 s-AML and 4 t-AML cases, respectively, diagnosed between 8/2005 and 5/2010. The median age of the cohort was 64.7 years. According to cytogenetically defined MRC criteria (Grimwade et al., Blood 2010) 63 patients of our cohort were assigned to the intermediate prognostic risk group and 13 to the poor risk group. Patients of the favorable prognostic risk group or those with “recurrent genetic abnormalities” according to WHO classification were excluded. In detail, 61 patients had a normal karyotype, 4 had other chromosomal aberrations and 11 had a complex aberrant karyotype. All coding exons of TET2, represented by 27 distinct PCR amplicons, were examined. For each amplicon a median of 643 reads was generated, thereby allowing a sensitive detection of variants, i.e. at a cut-off value of 10% 64 bidirectional reads were generated. In total, we observed 56 variances by this molecular mutation screening. After excluding 13 different polymorphisms and 1 silent mutation, 42 distinct mutations were detected in 26/76 (34%) patients. We identified 23 point mutations (14 missense and 9 nonsense; 55%) and 18 frameshift mutations (12 deletions, 5 duplications and 1 insertion; 43%). In 1/76 (2%) patients a splice site mutation was identified. The frameshift mutations ranged from 1 bp to 8 bp for deletions and 1 bp to 52 bp for duplications/insertions. The observed TET2 mutations were found to be heterogeneous and were spread over several exons. However, exons 3 and 11 could be identified as mutational hotspot regions, where 30/42 (71%) of the mutations were located. In exons 4, 5 and 9 only one mutation each was observed, respectively. No mutation was detected in exon 8. In detail, 25/42 (60%) mutations were located outside of the two conserved regions as described by Delhommeau et al. (N Engl J Med 2009), whereas 13 mutations (31%) were observed within the first conserved region spanning from codons 1134 to 1444 and 4 mutations (9%) were found within the second conserved region covering codons 1842 to 1921, respectively. 11/13 of the variances in the conserved regions were nonsense or frameshift mutations. Of the observed 42 mutations, only 8 had previously been described in the literature. The other 36 mutations represented here are novel. Generally, our results extend data known from the literature, i.e., the frequency of 34% of TET2 mutations in our AML cohort is higher than in previous publications, reporting a frequency between 12% and 20%. Concerning the mutational burden, the median range of the identified mutations was 44% of sequence reads carrying the mutation. Mutation load <20% was only observed in 2 cases (10% and 11%, respectively) and therefore would be below the detection level of Sanger sequencing. None of the detected TET2 mutations had a mutational burden of <10%. 15/26 (58%) patients carried more than one mutation and a mean of 1.6 mutations per patient was observed. In 2 patients two mutations within the same amplicon were detected and NGS was able to delineate subclones, i.e. in both cases approximately half of the reads carried either one mutation and only 1.7% and 2.3% of reads, respectively, harbored both mutations concomitantly. We further characterized this cohort according to mutations in NPM1 (n=73 cases investigated), FLT3-ITD (n=74), FLT3-TKD (n=64), CBL (n=76), and IDH1 (n=49). TET2 mutations were concomitantly observed with mutations in other molecular markers such as NPM1, FLT3-ITD, FLT3-TKD or CBL. Surprisingly, we found that TET2 mutations significantly excluded mutations in IDH1 (p=0.004). With respect to clinical data no difference in overall survival was monitored for AML patients that carried TET2 mutations (TET2-mutated 347 days vs. TET2 wild-type 294 days, n.s.). In conclusion, TET2 is a frequently mutated gene in AML. Due to the increasing complexity of observed molecular aberrations future studies will be essential to clarify the clinical relevance of TET2 mutations in AML and its prognostic and therapeutic role. Disclosures: Wille: MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

Abstract Non-coding (nc)RNAs, including circular (circ)RNAs, contribute to tumor development and progression. Several ncRNAs were shown to affect the onset, prognosis, and treatment of acute myeloid leukemia (AML) in the past years. The human Plasmacytoma Variant Translocation 1 (PVT1) gene maps on the long arm of chromosome 8 (8q24), in the same genomic region hosting MYC and encoding for 83 linear (PVT1, lncipedia.org) and 26 high-confidence circular isoforms (circPVT1, www.circbase.org). The most common isoform of circPVT1 is a product of back-splicing of 410 nt and contains the whole exon 2 of PVT1 in a closed loop-like structure (hsa_circ_0001821). The study aims to investigate the role of PVT1 isoforms and circPVT1 in AML. Firstly, we focused on the various PVT1 isoforms and their differential expression in leukemia. Fourteen out of the 83 linear isoforms are expressed in the hematopoietic tissues (lymph node and white blood cells, www.noncode.org), and 6 of them were detectable in AML cell lines, including the t(8;21) KASUMI-1 and the NPM1-mutated OCI-AML3 models, together with circPVT1. We designed two antisense-oligonucleotides (ASOs), mapping on common exonic region and targeting the linear isoforms expressed in OCI-AML3 and KASUMI-1 cells, and one ASO spanning the junction region of circPVT1. ASOs-mediated knockdown (KD) showed a relevant decrease of PVT1 signals, especially by ASO combination, and circPVT1 level using the specific ASO in both cell lines, under normoxia and hypoxia (1% O2). The downregulation led to a significant decrease in cell growth, but, interestingly, only circPVT1-KD induced apoptosis under both conditions in OCI-AML3. To further investigate the biological consequences of circPVT1-KD, we performed RNAseq assays. Data analysis was performed by pseudo alignment of paired-end reads to the human transcriptome, then counted with the Kallisto tool. Differential expression analysis of single isoforms was performed with the Sleuth tool on normalized transcript per million. We identified a core of 644 and 838 commonly regulated genes by circPVT1 in both cell lines under normoxia and hypoxia, respectively. Pathway analysis (performed by EnrichR) revealed that these genes are involved not only in the RNA regulatory pathways, as expected according to circRNA functions, but also in metabolic (e.g., KDM3A, GPI, NFKBA, RBM3, XBP1) and DNA damage response (e.g., PIDD1, MUC1, BCLAF1, BABAM2) pathways, opening a new scenario for synthetic lethality approaches. In conclusion, our findings show that silencing of circPVT1 or the predominantly expressed PVT1 isoforms dampens leukemia cell growth, indicating a role in AML pathogenesis, and suggest that targeting them may have therapeutic potentials in AML. Citation Format: Martina Ghetti, Antonella Padella, Eugenio Fonzi, Lorenzo Ledda, Andrea Ghelli Lusernadi Rorà, Matteo Paganelli, Doron Tolomeo, Clelia Tiziana Storlazzi, Giovanni Martinelli, Giorgia Simonetti. circPVT1 and linear PVT1 isoforms regulate cell growth, metabolic and DNA damage response related gene signatures in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1555.

Abstract Introduction: The CD30-positive cutaneous T-cell lymphoproliferative disorders (CD30-positive LPD) include lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large cell lymphoma (ALCL). Recurrent chromosomal translocations frequently underlie the pathogenesis of several hematologic malignancies and often define molecular subtypes with distinct biological behavior. The genetic events that contribute to the pathogenesis of CD30-positive LPD are largely unknown. Goal: The goal of our study was to identify chromosomal translocations that result in the generation of oncogenic chimeric gene fusions that underlie the pathogenesis of CD30-positive LPD. Methods and Results: Paired end whole transcriptome sequencing (RNAseq) analysis was performed on the cutaneous T-cell lymphoma (CTCL) derived cell lines MyLa and HH. Bioinformatic analysis revealed an interchromosomal fusion between NPM1 (5q35) and TYK2 (19p13). The NPM1-TYK2 fusion was represented by 766 paired-end reads spanning the fusion junction of exon 9 of NPM1 and exon 16 of TYK2. Bi-directional Sanger sequencing confirmed predicted gene fusion sequence at the RNA level. SYBR Green I-based quantitative RT-PCR assays revealed specific expression of the NPM1-TYK2 fusion transcript only in MyLa and not in other CTCL (n = 2) or T-cell lymphoma-derived cell lines (n = 7). To establish that the NPM1-TYK2 gene fusion arose from a translocation event at the DNA level, we performed long range PCR which yielded a 1kb product containing the breakpoints and junctions in NPM1 (chr5:170,832,813) and TYK2 (chr19:10,469,817) introns that lead to juxtaposition of these genes by translocation. The fusion is predicted to encode an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. To determine if translocations targeting TYK2 were recurrent, we employed two independent fluorescence in situ hybridization (FISH) based assays, one using a TYK2 break-apart probe strategy to identify translocations targeting TYK2 and a second approach using an NPM1-TYK2 fusion probe strategy to specifically identify the NPM1-TYK2 translocations. These probes were used to screen patient-derived biopsies of CD30-positive LPD, CTCL and other T-cell neoplasms. FISH analyses revealed TYK2 translocations in 5 of 29 (17.2%) primary cases of CD30-positive LPDs (3/15 LYP; 2/14 cutaneous ALCLs) and was absent in other CTCL subtype (Mycosis fungoides, n = 44) and mature T-cell neoplasms (n = 107). Out of these 5 positive cases, one LYP case had an NPM1-TYK2translocation. Since TYK2 is a member of Jak family kinases that are integral components of the JAK-STAT signaling pathway, we investigated the downstream effects of NPM1-TYK2 fusion expression on TYK2 kinase activity and STAT protein pathway activation. Western blotting in MyLa, other cutaneous and mature T-cell lymphoma cell lines revealed hyperactivation of endogenous TYK2 kinase enzyme (pTYK2 levels) only in MyLa. Further, MyLa cells showed activation of downstream STAT signaling pathway proteins (pSTAT1, pSTAT3 and pSTAT5). Ectopic expression of NPM1-TYK2 wild type fusion gene in HEK293FT cells resulted in hyperactivation of TYK2 kinase enzyme and activation of STAT proteins, while a kinase-defective mutant NPM1-TYK2 K462R lacked TYK2 and STAT activity. Transcriptional activation assays for STAT proteins (STAT1, STAT3 and STAT5), showed more than two fold (P<0.01) elevation of reporter activity of the aforementioned STATs in NPM1-TYK2 wild type fusion protein expressing but not in kinase-defective mutant expressing cells. Silencing of TYK2 using a lentivirus-based shRNA approach resulted in decreased proliferation (P<0.01, 2.2 fold) of MyLa cells suggesting that NPM1-TYK2is an oncogenic-driver alteration. Conclusions: Our study demonstrates for the first time recurrent TYK2 gene translocations in CD30-positive LPD or in any form of primary cancer. We identify NPM1 as one of the gene-fusion partners of TYK2 and provide functional support for NPM1-TYK2 in mediating activation of STAT signaling to promote cell proliferation. Inactivation of TYK2 significantly diminishes proliferation, suggesting that TYK2 is an oncogenic driver kinase in a subset of CD30-positive LPD. Finally, our results raise the possibility that TYK2 may be a novel therapeutic target in a subset of CD30-positive LPDs. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Next generation sequencing (NGS) applications have recently identified various recurrent kinase and cytokine receptor rearrangements in a subgroup of B-progenitor acute lymphoblastic leukemia (BPC-ALL) that is characterized by a Ph-like gene expression signature. Implementation of whole genome applications into diagnostics have so far been hampered by high costs and long turnover times. Therefore we developed a custom-made NGS based capture panel without the need for gene specific PCR amplification steps. With a rapid turnover time, pooling of multiple samples and standardized bioinformatics, this approach can be implemented in molecular diagnostics of BCP-ALL to identify patients who may benefit from an add-on therapy with specific tyrosine kinase inhibitors. Moreover, genomic breakpoints can be used as an additional patient specific target for minimal residual disease (MRD) monitoring. Here we report a feasible approach for the identification of kinase and kinase-dependent pathway aberrations in BPC-ALL utilizing a customized sequence enrichment capture panel for NGS. Patients and Methods: Ninety three samples from BCP-ALL patients with detectable minimal residual disease (MRD) levels (≥ 5x10-4) after induction therapy from COALL studies 07-03 or 08-09 were applied to the NGS custom panel. Genomic capture probes were specific for exonic and complete intronic regions of ABL1, JAK2, PDGFRB, CRLF2, EPOR, and for the IgH-JH region on chromosome 14. JAK1, JAK3, IKZF1 and SH2B3 were analysed for exonic mutations only. These predefined genomic regions from recurrently mutated or rearranged kinase and cytokine receptor genes were enriched from 50 ng of initial genomic DNA without gene specific PCR amplification steps, followed by paired end sequencing of the captured genomic fragments (Nextera Rapid Capture Custom enrichment protocol with paired end sequencing on a MiSeq benchtop sequencing platform; Illumina). Six positive controls with known rearrangements (BCR-ABL1 and ID4-JH) were included to verify this method. Results: All breakpoints from 6 positive controls were successfully identified and verified by patient specific amplification of the genomic fusion sites. ABL1 and JH specific capture probes allowed the "fishing" of the genomic fusion partner gene, here BCR and ID4, by paired end sequencing of overlapping, breakpoint spanning reads. In addition we identified a genomic breakpoint for CRLF2, PDGFRB, EPOR, ABL1 and JAK2 in 19/93 BCP-ALL patient samples. One additional patient showed a SH2B3 5 bp insertion in exon 5 that leads to a premature stop codon. The 11 patients with either EBF1-PDGFRB (4), EPOR-JH (4), or CRLF2-JH (3) rearrangements showed the highest levels of MRD (≥ 1x10-2) at the end of induction. MRD monitoring using the genomic breakpoint in 3 cases showed highly concordant data to our standardized IgH/TCR quantification assays. For the 3 patients tested so far, our breakpoint specific MRD assays clearly showed the association of these rearrangements with the dominant leukemic clone. Discussion: In summary, we identified kinase- or cytokine receptor rearrangements in 20/93 patients analyzed with our NGS based custom enrichment panel. The majority of patients had high MRD level at EOI therapy and an age of onset ≥ 10 years. Rapid identification of kinase- or cytokine receptor rearrangements in BCP-ALL is necessary for timely therapeutic intervention with target specific tyrosine kinase inhibitors. Short turnaround times of less than 2 weeks and processing of multiple samples followed by a standardized bioinformatical workflow allows the identification of unknown fusions partners from frequently rearranged kinase- and cytokine receptor genes, providing the exact breakpoint location, irrespective of the gene expression signature. MRD guided NGS panel analysis is a feasible strategy for identification of patients with a high risk of therapeutic failure or relapse suitable for targeted TKI intervention. Table 1.Age at DxFusionMRD at EOI13EBF1-PDGFRB1,0E+009EBF1-PDGFRB9,0E-0113EBF1-PDGFRB3,0E-019EBF1-PDGFRB7,0E-0216EPOR-IGH6,0E-017EPOR-IGH3,0E-0116EPOR-IGH1,0E-0118EPOR-IGH9,0E-0211CRLF2-IGH9,0E-0211CRLF2-IGH4,7E-0216CRLF2-IGH4,0E-0215PAX5-JAK27,0E-0411RCSD1-ABL15,0E-0210SH2B3-INS3,0E-0310PAR15,0E-0110PAR17,0E-021PAR11,8E-025PAR17,0E-038PAR12,0E-032,5PAR11,5E-03 Disclosures No relevant conflicts of interest to declare.

AbstractSingle-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample.

Duchenne muscular dystrophy (DMD), one of the most common progressive and severely disabling neuromuscular diseases in children, can be largely attributed to the loss of function of the DMD gene on chromosome Xp21.2-p21.1. This paper describes the case of a 10-year-old boy diagnosed with DMD. Whole exome sequencing confirmed the hypothesized large partial exonic deletion of c.7310-11543_7359del (chrX:g.31792260_31803852del) spanning exon 51 and intron 50 in DMD. This large deletion was verified to be de novo by PCR, and the two breakpoints were further confirmed by Sanger sequencing and long-read whole-genome sequencing. Notably, this partial exonic deletion was the only complex variation in the deep intron regions or intron–exon junction regions in DMD. In addition, the case study demonstrates the clinical importance of using multiple molecular genetic testing methods for the diagnosis of rare diseases.

BRAF is a widely studied oncogene and its functions are well characterized in several cellular contests and diseases. However the regulation of the expression of BRAF is mostly unknown. With the aim to understand the post-transcriptional regulation of BRAF, we performed 3’RACE in A375 melanoma cells and we found 2 different 3’UTRs: the one commonly reported in many data bases (Reference) and a new one that is only predicted (X1). The two 3’UTRs are completely different in sequence and length (120nt vs 1350nt). Furthermore, they are transcribed from exon 18 or thanks to an alternative splicing event occurring between exon 18 and a newly discovered exon 19 (X1). By using Real Time PCR, we confirmed the expression of both transcripts in melanoma and non-melanoma cell lines. Moreover, using RNA-SEQ data available at TCGA, we showed the co-expression of Reference and X1 BRAF transcripts also in human biopsies. Due to our discovery that BRAF exists in at least 2 different transcript variants, we decided to investigate further the expression of all the BRAF isoforms reported in NCBI and Ensembl. To do so, we took advantage of the RNA-seq data of more than 4,800 patients belonging to 9 different cancer types. We show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1 and BRAF-X2) that differ in the last part of their open reading frames, as well as in the length (BRAF-ref: 76nt; BRAF-X1 and X2: up to 7kb) and in the sequence of their 3’UTRs. In melanoma cells, the X1 isoform is expressed at the highest level, while the most prevalent among the three isoforms varies from one cancer type to another. Moreover, the relative abundance among the three BRAF isoforms is maintained in melanoma cells with acquired resistance to BRAF and MEK inhibitors driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. Besides their 3’UTRs, also the very last part of the coding sequences differ among the three isoforms. By immunoprecipitation of BRAF in A375 cells and subsequently Mass-SPEC analysis, we revealed the existence of Reference and X1 proteins which are expressed at similar levels, while X2 is not detectable because quicky degraded by the proteasome. Furthermore functional studies show that the two proteins account together for BRAF activities both in vitro and in vivo. Given the differences in length and sequence between the reference and the X1 3’UTR, we hypothesized that the two isoforms undergo different regulation mediated by RNA-binding proteins or non-coding RNAs. We focused on post-transcriptional regulation by microRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the expression of target messenger RNAs (mRNAs) and for this reason play a key role in virtually all cellular processes. In spite of the availability of several prediction algorithms, the identification of specific miRNA-target interactions remains a challenge. In order to overcome this problem we developed an innovative method, called miR-CATCH v2.0, for the high-throughput identification of microRNAs that bind a target transcript. The protocol is based on the affinity purification of the target mRNA and bound miRNAs by using two different pools of 3’biotinylated anti-sense DNA probes (ODD and EVEN). We designed 12 probes (6 ODD probes and 6 EVEN probes) for the purification of X1-3’UTR-miRNAs complexes and we performed three separate and independent captures in A375 metastatic melanoma cells. MicroRNAs were identified through small RNA-sequencing and the top-scoring miRNAs that resulted consistently enriched in all the captures will be validated in vitro and in vivo experiments.