PMC9798314_01
Discovery of the index case. (A) Principal component analysis revealed one relapsed acute myeloid leukemia (AML) case (indicated by the red arrow) separate from other AML cases, but adjacent to the cluster of acute promyelocytic leukemia (APL) cases. (B) Morphology of the bone marrow (BM) smear at relapse, Wright's stain.
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BM morphology examination showed that 60% of nucleated cells were blast cells with abundant purple granules, irregular nuclei, and cytoplasmatic budding, while negative for Auer rods ( Figure 1B ).
TTMV integration structure of our case. (A) Alignment of the whole transcript sequencing (WTS) data in integrative genomics viewer (IGV) revealed abundant RARA fusion transcripts with a 5' extension from RARA exon 3, which is not homologous to any human sequence. (B) Schematic representation of the fusion between the TTMV sequence and RARA exon 3. The chimeric transcript includes TTMV open reading frame 2 (ORF2) and an upstream conserved untranslated region (UTR), in-frame with RARA exon 3. (C) Sanger sequencing confirmed the existence of the TTMV::RARA fusion transcript. (D) The predicted structure of the TTMV::RARA fusion protein. The blue arrow indicates the highly conserved TTMV ORF2 motif. The red arrow at position 85 indicates the fusion site. (E) Whole genome sequencing (WGS) revealed the insertion was located at chr17: 40332265 (hg38) in RARA intron 2. (F) Sanger sequencing confirmed the two genomic junction sequences. (G) Schematic representation of the location and structure of the integrated TTMV sequence. (H) TTMV insertion characteristics in the 4 cases reported to date. The red arrows show the insertion positions of TTMV sequence in RARA intron 2 (hg38). The black rectangles show the integrated TTMV sequences, and the yellow blocks within them show the nucleotides expressed in the TTMV::RARA fusion transcript.
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Figure 2A ). Further analysis revealed the exogenous sequence started with 58 nucleotides that aligned to the untranslated region (UTR) of TTMV, followed by 256 nucleotides starting with a start codon (ATG) and revealed a significant alignment to different TTMV isolates (83% coverage, 93% to 94% identity), in-frame with the full TTMV::RARA fusion transcript ( Figures 2B, C ). TTMV::RARA fusion transcript was predicted to encode a fusion protein containing 488 amino acids ( Figure 2D ). The putative N terminal 85 polypeptide sequence showed 54% identity and 45% coverage to TTMV open reading frame 2 (ORF2) and displayed the conserved motif WX7HX3CXCX5H, which were shared among all anelloviruses. The RARA part of the fusion protein was consistent with PML::RARA and all other RARA fusions. TTMV genome ( Figures 2E, F ). There were 750 nucleotides and 87 nucleotides derived from TTMV genomic sequences upstream and downstream of the TTMV UTR region, respectively. Also, there were 13 nucleotides not belonging to any known TTMV isolates downstream of TTMV ORF2, all of which were removed during splicing ( Figure 2G ). TTVs. The last four bases (AGCC) of RARA intron 2 sequence 5' to the insertion site were the same as the four 3' terminal bases of the inserted sequence, suggesting there might be a microhomologous recombination mechanism that mediated the viral sequence integration ( Figure 2G ).