Towards a better understanding of the low recall of insertion variants with short-read based variant callers

Since 2009, numerous tools have been developed to detect structural variants (SVs) using short read technologies. Insertions >50 bp are one of the hardest type to discover and are drastically underrepresented in gold standard variant callsets. The advent of long read technologies has completely changed the situation. In 2019, two independent cross technologies studies have published the most complete variant callsets with sequence resolved insertions in human individuals. Among the reported insertions, only 17 to 37% could be discovered with short-read based tools. In this work, we performed an in-depth analysis of these unprecedented insertion callsets in order to investigate the causes of such failures. We have first established a precise classification of insertion variants according to four layers of characterization: the nature and size of the inserted sequence, the genomic context of the insertion site and the breakpoint junction complexity. Because these levels are intertwined, we then used simulations to characterize the impact of each complexity factor on the recall of several SV callers. Simulations showed that the most impacting factor was the insertion type rather than the genomic context, with various difficulties being handled differently among the tested SV callers, and they highlighted the lack of sequence resolution for most insertion calls. Our results explain the low recall by pointing out several difficulty factors among the observed insertion features and provide avenues for improving SV caller algorithms and their [email protected]

Identifying NAHR mechanism between two distinct Alu elements through breakpoint junction mapping by NGS

Background: Genomic rearrangements encompass deletions, duplications, inversions, insertions and translocations and may be the cause of several genetic diseases. One of the most frequent mechanisms that generates these rearrangements is the Non-Allelic Homologous Recombination (NAHR). They are caused by a misalignment between regions of high level of similarity, like Low Copy Repeats (LCRs) and Alu sequences. We aimed to sequence the breakpoint of a patient with a single deletion on chromosome 22q13.2 in order to understand the genomic structure of the region involved as well as elucidate the mechanism behind this rearrangement. Investigating breakpoints are of the utmost importance in the understanding the influence of the genomic architecture in clinical assays. Results: We flanked the breakpoint detected by array and then we captured the regions using Illumina Nextera Rapid Capture Custom to sequence with Illumina MiSeq. We found a chimeric read on Chr22:41,026,090, setting a 624,688 bp deletion on Chr22:41,026,112-41,650,780 (hg19). This deletion merges the intronic region of MKL1 and RANGAP1 genes, on two different Alu sequences ( AluSx and AluY, respectively ). Conclusions: The sequence of the breakpoint reveals that Alu elements are an important characteristic of the human genome on generating rearrangements.

RNAi technology targeting the FGFR3-TACC3 fusion breakpoint: an opportunity for precision medicine

Background Fusion genes form as a result of abnormal chromosomal rearrangements linking previously separate genes into one transcript. The FGFR3-TACC3 fusion gene (F3-T3) has been shown to drive gliomagenesis in glioblastoma (GBM), a cancer that is notoriously resistant to therapy. However, successful targeting of F3-T3 via small molecular inhibitors has not revealed robust therapeutic responses, and specific targeting of F3-T3 has not been achieved heretofore. Here, we demonstrate that depleting F3-T3 using custom siRNA to the fusion breakpoint junction results in successful inhibition of F3-T3+ GBMs, and that exosomes can successfully deliver these siRNAs. Methods We engineered 10 unique siRNAs (iF3T3) that specifically spanned the most common F3-T3 breakpoint with varying degrees of overlap, and assayed depletion by qPCR and immunoblotting. Cell viability assays were performed. Mesenchymal stem cell–derived exosomes (UC-MSC) were electroporated with iF3T3, added to cells, and F3-T3 depletion measured by qPCR. Results We verified that depleting F3-T3 using shRNA to FGFR3 resulted in decreased cell viability and improved survival in glioma-bearing mice. We then demonstrated that 7/10 iF3T3 depleted F3-T3, and importantly, did not affect levels of wild-type (WT) FGFR3 or TACC3. iF3T3 decreased cell viability in both F3T3+ GBM and bladder cancer cell lines. UC-MSC exosomes successfully delivered iF3T3 in vitro, resulting in F3-T3 depletion. Conclusion Targeting F3-T3 using siRNAs specific to the fusion breakpoint is capable of eradicating F3T3+ cancers without toxicity related to inhibition of WT FGFR3 or TACC3, and UC-MSC exosomes may be a plausible vehicle to deliver iF3T3.

Bruton's Tyrosine Kinase (BTK) Is a Novel KMT2A Partner Gene

Histone-lysine N-methyltransferase 2A gene (KMT2A) rearrangements are common genetic events in acute leukemia. They more frequent in infants and are found in 30-45% of acute myeloid leukemias (AML). They demonstrate great molecular heterogeneity with more than 90 partner genes and multiple KMT2A breakpoints involved (Meyer et al., 2018). These characteristics challenge both initial molecular diagnostics and MRD monitoring in ALs with KMT2A rearranged. As the accurate detection of all KMT2A-r types is crucial in order to correct patients' therapy and risk groups' definition, we aim to fully characterize KMT2A rearrangements within our cohort of patients using various techniques. Our recent study demonstrates a novel KMT2A partner gene - Bruton's tyrosine kinase (BTK), in a case of childhood AML. The patient is 9 m.o. girl who presented with WBC 69,3*109/L and hepatosplenomegaly. Morphological, immunochemical and immunological examination revealed acute monocytic leukemia. Bone marrow aspirates were analyzed by G-banded karyotyping, FISH with KMT2A breakapart probe. The karyotype was 45,X,der(X?)t(X;11)(q22.1;q23.3),del(11)(q14),-20[7] with 95% KMT2A-rearranged nuclei. Real-time RT-PCR for 8 most common KMT2A rearrangements screening was negative. Targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA) followed by high-throughput sequencing identified novel fusion transcript KMT2A-BTK with exon 9-exon 2 breakpoint junction. Sanger sequencing was used for validation. The achieved data on KMT2A translocation partner and breakpoint location was used for subsequent patient-specific MRD monitoring. The patient was treated according to AML-MRD-2018 local protocol and achieved complete MRD-negative remission after induction course. Then, a severe myelodepression developed after consolidation therapy, and the patient died due to infectious complications. BTK gene is located at Xq22.1 and encodes for Bruton's tyrosine kinase protein (Hashimoto et al., 1996), which is a cytoplasmic non-receptor tyrosine kinase essential for B-cell maturation and development (Shinners et al., 2007). BTK deficiency is strongly associated with X-linked agammaglobulinemia, where 80-90% of patients express either truncated or misfolded or mutated BTK protein (Valiaho, Smith, & Vihinen, 2006). BTK is also implemented in B-cell acute lymphoblastic leukemia, where it often has a deleted or a truncated kinase domain (Feldhahn et al., 2005). The opposite situation was reported for AML in several studies showing increased BTK activity (Gu et al., 2011; Tomasson et al., 2008). The presented t(X;11)(q22.1;q23.3) childhood AML is to our knowledge the first case of BTK translocations in hematologic malignancies, and its value for leukemogenesis is to be further investigated. However, as the breakpoint junction is located in BTK intron 1, upstream of its coding region (the translation starts in exon 2), we can speculate that BTK's structure itself is not committed and its activity is increased due to upregulated expression together with KMT2A. Therefore, the novel fusion KMT2A-BTK was for the first time revealed during the molecular profiling of KMT2A-rearranged AML in Russian Federation. The work was supported by RFBR grant №17-29-06052. Disclosures No relevant conflicts of interest to declare.

Unique Familial MLL-rearranged Precursor B Cell Infant Acute Lymphoblastic Leukemia (ALL) in Non-Twin Siblings

Abstract 2417 Leukemia is the commonest malignancy in infants, the most frequently occurring form of which is infant ALL. When ALL occurs in infants the disease is clinically aggressive and associated with poor outcome. MLL gene rearrangements producing transforming fusion oncoproteins are found in 75% of infant ALL and they are adverse prognostic factors. Infant ALL has never occurred in families except in monozygous twins, where concordance in leukemia occurrence is nearly 100%. Identical but non-germline genomic breakpoint junction sequences have pointed to an in utero origin of MLL gene rearrangements in these twin cases, where it is believed that metastasis of cells with the rearrangement occurs from one twin to the other via the placental circulation. Here we describe two highly novel siblings both deceased from precursor B cell infant ALL (ages at diagnosis: proband 160 d, sibling 121 d). Remarkably, the second of these two decedents is survived by a now 3 year-old monozygous twin who is as yet unaffected. MLLrearrangements in the leukemia blasts of both affected siblings were characterized by conventional cytogenetics and/or FISH, M-FISH, high resolution Illumina 610K Bead Chip SNP array and Southern blot analysis. MLL genomic breakpoint junction sequences and fusion transcripts were defined using panhandle PCR approaches, PCR with gene-specific primers and reverse transcriptase PCR. The twins were confirmed to be monozygous using the genotype calls from SNP array analysis of the peripheral blood and bone marrow of the unaffected and affected twins, respectively. Quantitative real-time PCR analysis of leukemia DNA was used to determine the allele specific sequences of the NQO1 (NADPH quinone oxidorecutase 1) gene, an inactivating polymorphism in which previously was implicated as a risk factor for MLL-rearranged infant ALL. The complex karyotype in the leukemia cells of the proband was 46, XX, der(2) t(2;3) (q3?;?), der(3) ?t(3;4;11), del(4) (q21), der(11) ?del(11) (p11.2) t(3;11) (?;q23).ish der(3) (5'MLL+), der(11) (3'MLL+) [14]/46, XX[8], suggesting extensive damage to the genome. Consistent with a three-way t(3;4;11) translocation, two alternately spliced 5'-MLL-MLLT2(AF-4)-3' fusion transcripts were identified, indicating disruption of the chromosome band 4q21 partner gene MLLT2. Also consistent with the three-way translocation, reverse panhandle PCR detected a 5'-partner-MLL-3' genomic breakpoint junction fusing 3' MLL to the upstream region of a highly novel chromosome 3 gene encoding a nucleotidyltransferase fold protein C3ORF31 (Accession no. NM_138807; Kuchta, 2009). Unlike in the proband, the ALL of the affected twin exhibited a simple t(4;11)(q21;q23) translocation, the reciprocal genomic breakpoint junctions of which fusing MLL and MLLT2 also have been characterized. Similar to non-familial infant ALL, the genomic breakpoint junctions in both infants contained sequence features of nonhomologous end joining DNA repair. The different MLL gene rearrangements in the leukemia cells in the affected siblings indicate that the translocations were not hereditary. Even though the NQO1 inactivating polymorphism is one genetic risk factor for infant ALL, the NQO1 genotype was wild-type in both affected siblings. Further studies in this uniquely afflicted family with two siblings who succumbed to infant ALL and a monozygous twin of one of the decedents surviving beyond infancy unaffected, will provide a one-time opportunity to capture novel mutations predisposing to the development of, and cooperating with, MLL translocations in infant ALL. The MLLT2 involvement in both cases has even further implications for the knowledge to be gained because the MLL-MLLT2 rearrangement occurs in 50% of infant ALL and adversely impacts outcome. Disclosures: Felix: Children's Hospital of Philadelphia: Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia - U.S. Patent # 6,368,791 issued April 9, 2002.

Cryptic MLL, AF10, ARMC3 Rearrangement in Secondary AML Creates ARMC3-MLL Fusion That Disrupts Gene Encoding Armadillo Repeats Similar to Major Cancer Genes.

Introduction: Complex rearrangements are required to form 5′-MLL-AF10-3′ translocation breakpoint junctions because the 5′ to 3′ orientation of AF10 at band 10p12 is telomere to centromere. Previously we discovered a cryptic MLL translocation and 5′-MLL-AF10-3′ fusion transcripts in a case of secondary FAB M4 AML with a normal karyotype, which occurred after rhadomyosarcoma treatment with etoposide (Megonigal 2000). Here we characterized the genomic breakpoint junctions of the translocation, which not only proved to be a 3-way rearrangement, but also to involve a novel partner gene. Methods: BamHI reverse panhandle PCR was used to clone the unknown 5′-partner gene-MLL-3′ genomic breakpoint junction. The corresponding fusion transcript was obtained by RT-PCR. The 5′-MLL-AF10-3′ genomic breakpoint junction was obtained by PCR with gene specific primers. Results: Consistent with the smaller of two BamHI MLL bcr rearrangements on the Southern blot, BamHI reverse panhandle PCR gave a 4090 bp product, with the ARMC3 (armadillo repeat containing 3) gene from band 10p12 at the 5′ side fused to the 3′ portion of the MLL bcr. The ARMC3 breakpoint was position 1901 or 1902 relative to the start of intron 17. The MLL bcr breakpoint at position 6473 or 6474 in intron 8, was 5′ to the secondary leukemia MLL translocation breakpoint hotspot. Identical C residues at the ARMC3 and MLL breakpoints precluded more precise breakpoint determinations and suggested NHEJ DNA repair typical of other MLL genomic breakpoint junctions. The novel rearrangement produced an in-frame 5′-ARMC3-MLL-3′ transcript with ARMC3 exon 17 and MLL exon 9 at the point of fusion, in addition to two alternatively spliced 5′-MLL-AF10-3′ transcripts joining MLL exon 7 or MLL exon 8 to AF10 exon 10. Southern blot analysis, restriction mapping, the 5′-MLL-AF10-3′ fusion transcripts and the 5′-ARMC3-MLL-3′ genomic breakpoint junction informed gene-specific primers for PCR-based cloning of the 5′-MLL-AF10-3′ genomic breakpoint junction. MLL bcr position 6202, 6203, 6204, 6205, 6206 or 6207 in intron 8 was fused to position 2737, 2738, 2739, 2740, 2741 or 2742 relative to the start of AF10 intron 9, and 5′-ATTAG-3′ sequences were present at the breakpoints in both genes. Comparison of the 5′-MLL-AF10-3′ and 5′-ARMC3-MLL-3′ genomic breakpoint junctions indicated that 265 to 271 bases from MLL were deleted during the translocation. Conclusions: Since ARMC3 is adjacent and centromeric to AF10 at band 10p12, and has the same 5′ to 3′ orientation from telomere to centromere, these results suggest that the complex 3-way rearrangement occurred by splitting of band 11q23 and insertion of band 10p12 material containing the 3′ portion of AF10 through to the 5′ portion of ARMC3 into the MLL bcr. Formation of the 5′-MLL-AF10-3′ and 5′-ARMC3-MLL-3′ rearrangements on the der(11) chromosome in this manner is consistent with the formation of 5′-MLL-AF10-3′ breakpoint junctions via complex translocations. Additional studies are in progress to determine whether a 5′-ARMC3-AF10-3′ genomic breakpoint junction was created on the der(10) chromosome. The uncharacterized ARMC3 protein is of interest because catenin proteins (eg. β-catenin), plakophilins and the tumor suppressor APC contain Arm repeats. This raises the possibility of a potential contribution of the predicted ARMC3-MLL fusion protein in the genesis of the AML.