Targeted Next Generation Sequencing Reveals a Third Breakpoint Cluster Region and New Partner Genes in the KMT2A Recombinome

Chromosomal rearrangements of the KMT2A gene are associated with acute leukemias and myelodysplastic syndromes. The large number of known KMT2A fusions (>100) renders a precise diagnosis a demanding task. More than 50% of all KMT2A partner genes have been analyzed at the DCAL, including the novel partner genes BCAS4, FAM13A, RANBP3, and STK4. Even though all KMT2A rearrangements are associated with high-risk acute leukemia, the outcome (poor or very poor) is influenced by the partner gene. So far, we have analyzed more than 3,200 patients positive for a KMT2A rearrangement. The breakpoints of these cases are located mainly in the major breakpoint cluster region (bcr1) and to a small extent in the recently described minor bcr (bcr2). A small number of breakpoints were also found outside of these two bcrs. Most of these patients were analyzed by long distance inverse (LDI)- or multiplex-PCR which only cover bcr1. More recently, we used targeted KMT2A-NGS with whole gene coverage in over 450 patients, which was initially applied selectively in patients negative by LDI- and multiplex-PCR and then used more widely. Within the KMT2A-NGS group, 410 patients had bcr1 breakpoints mainly between the KMT2A exons 7 and 13, while 46 patients bcr2 breakpoints mainly between exons 20 and 24. Of note, five patients had their breakpoint outside of these two bcrs: three of them within intron 2 and no functional KMT2A rearrangement; the other two within intron 35 and intron 36, fusing almost the whole KMT2A gene in frame to the respective partner genes ARHGEF12 and MLLT4. These two breakpoints may define a third and rare bcr (bcr3), although further cases are needed to support this hypothesis. Interestingly, 70 patients displayed a 3'-KMT2A deletion, indicating that the number of terminal deletions is higher than described previously. Two patients had a 5'-KMT2A deletion. All deletions started or ended in bcr1 and bcr2. We also observed a striking difference in the distribution of partner genes between bcr1 and bcr2. The most frequent translocation partners fused to bcr1 sites are transcription factors, while the partner genes linked to bcr2 sites generally code for cytosolic proteins. In bcr1, the 4 most frequent partner genes AFF1, MLLT3, MLLT1, and MLLT10, found in 80% of cases, all code for transcription factors that are part of the super elongation complex (SEC). These fusions therefore all lead to disruption of the hematopoietic lineage commitment. In contrast in bcr2, 3 partner genes USP2, MLLT4, and USP8 account for 85% of the cases. USP2 and USP8 are ubiquitin specific peptidases involved in cell signaling and exclusively fused to bcr2 in KMT2A. While MLLT4 is found as a partner in bcr1, bcr2 and bcr3 fusions; unlike other recurrent KMT2A partners linked to bcr1, it is not a transcription factor and it exerts oncogenic potential via dimerization like other cytosolic partners. We hypothesize that the oncogenic properties of USP2 and USP8 are dependent on dimerization like MLLT4 and that the most frequent fusions involving at different bcrs favor different oncogenic mechanisms: bcr1 transactivation and bcr2 dimerization. Further studies are needed to explain why USP2 and USP8 are exclusively associated with bcr2, and why the most frequent partner genes AFF1 and MLLT3 of the bcr1 are less frequent in bcr2. In conclusion, targeted NGS combined with bioinformatic analysis has expanded our knowledge of the KMT2A recombinome to include more fusion partners and has generated new hypotheses for future research on oncogenic mechanisms. Disclosures No relevant conflicts of interest to declare.

Image Splicing Location Based on Illumination Maps and Cluster Region Proposal Network

Splicing is the most common operation in image forgery, where the tampered background regions are imported from different images. Illumination maps are inherent attribute of images and provide significant clues when searching for splicing locations. This paper proposes an end-to-end dual-stream network for splicing location, where the illumination stream, which includes Grey-Edge (GE) and Inverse-Intensity Chromaticity (IIC), extract the inconsistent features, and the image stream extracts the global unnatural tampered features. The dual-stream feature in our network is fused through Multiple Feature Pyramid Network (MFPN), which contains richer context information. Finally, a Cluster Region Proposal Network (C-RPN) with spatial attention and an adaptive cluster anchor are proposed to generate potential tampered regions with greater retention of location information. Extensive experiments, which were evaluated on the NIST16 and CASIA standard datasets, show that our proposed algorithm is superior to some state-of-the-art algorithms, because it achieves accurate tampered locations at the pixel level, and has great robustness in post-processing operations, such as noise, blur and JPEG recompression.

The role of reciprocal fusions in MLL-r acute leukemia: studying the chromosomal translocation t(6;11)

Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9–10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21–23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the “Massive Analyses of cDNA Ends” (MACE-Seq) technology, an established 3′-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.

Deletion of 2 amino acids in IHH in a Japanese family with brachydactyly type A1

Background Brachydactyly type A1 (BDA1) is an autosomal dominant disorder characterized by uniform shortening of the middle phalanges in all digits. It is associated with variants in the Indian Hedgehog (IHH) gene, which plays a key role in endochondral ossification. To date, heterozygous pathogenic IHH variants involving several codons, which are restricted to a specific region of the N-terminal active fragment of IHH, have been reported. The purpose of this study was to identify the pathogenic variant in a Japanese family with BDA1 and to evaluate its pathogenesis with regard to previous reports. Methods The proband, a 9-year-old boy, his siblings, and his father had shortened digits and a short stature of variable severity. Based on physical examinations, radiographic findings and family history, they were diagnosed with BDA1. This family is the first case of an isolated malformation in Japan. Sanger sequencing of IHH was performed on these individuals and on the proband’s unaffected mother. The significance of the variants was assessed using three-dimensional analysis methods. Results Sanger sequencing showed a novel IHH heterozygous variant, NM_002181.4:c.544_549delTCAAAG(p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del].. These two residues are located outside the cluster region considered a hotspot of pathogenic variants. Three-dimensional modelling showed that S182 and K183 are located on the same surface as other residues associated with BDA1. Analysis of residue interactions across the interface between IHH and its interacting receptor protein revealed the presence of hydrogen bonds between them. Conclusions We report a novel variant, NM_002181.4:c.544_549delTCAAAG (p.Ser182Lys183del) [NC_000002.12:g.219057461_219057466del] in a Japanese family with BDA1. Indeed, neither variations in codons 182 or 183 nor with such two-amino-acid deletions in IHH have been reported previously. Although these two residues are located outside the cluster region considered a hotspot of pathogenic variants, we speculate that this variant causes BDA1 through impaired interactions between IHH and target receptor proteins in the same manner as other pathogenic variants located in the cluster region. This report expands the genetic spectrum of BDA1.