PMC9213071_01
(A) A significantly increased eosinophil ratio was shown on a bone marrow (BM) smear (Wright-Giemsa stained, x 100). (B) Significantly increased eosinophilia was observed on a peripheral blood (PB) smear (x 100), the arrows delineated eosinophils. (C) Karyotypic analysis of BM showed the translocation of t(5; 12) (q31; p13) (delineated by arrows). (D) RNA-sequencing analysis indicated the IL3-ETV6 fusion. (E) Circos plot displaying the interconnectivity between IL3 and ETV6. (F) Amplified IL3-ETV6 transcripts by RT-PCR. Control: water. The predicted product was 300bp, and the patient's PCR product was indicated by red arrow. (G) Sanger sequencing of the PCR product (IL3-ETV6). (H)
IL3 mRNA was obviously more highly expressed than in other eosinophilia patients with normal karyotypes. Control: CEL-NOS patients with normal karyotype, FPKM: fragments per kilobase of transcript sequence per millions base pairs sequenced. (I) Fluctuation of the patient's peripheral white blood cell count (WBC, x109/L) and eosinophil ratio (EOS, %) during treatment.
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Bone marrow (BM) and peripheral blood (PB) smears showed a high ratio of eosinophils (26% in BM, 47% in PB) ( Figures 1A, B ), and the ratio of granulocytes: erythrocytes in BM was 13.3:1 (1.28-5.95:1). The karyotype analysis revealed 46, XY, t(5;12) (q31; p13) ( Figure 1C ). IL3-ETV6 ( Figures 1D, E ). In this translocation, exons 3 to 5 of IL3 on chromosome 5 were spliced, inverted and fused with exons 3 to 8 of ETV6 on chromosome 12. In addition, a transcript of GATA2-SOCS2 (exon 1 of GATA2 (5'UTR) fusing with exon 2 to 3 of SOCS2 (5'UTR)) was also detected ( Supplementary Figure 1 ). Reverse transcription PCR (RT-PCR) was performed to confirm the IL3-ETV6 fusion by the following primers: forward (at IL3 intron 2-3), 5'AAATCA CAGAGACCCCAGC3' and reverse (at ETV6 exon 3), 5'AAGG AGTTCATAGAGCACATCA3', and a product of approximately 300 bp was observed as we predicted ( Figure 1F ). Sanger sequencing analysis of this product showed the detailed breakpoints and fusion segments ( Figure 1G ). Compared with other CEL-NOS patients with normal karyotype, significantly higher (more than 700-fold change) fragments per kilobase of transcript sequence per million base pairs of IL3 mRNA in this patient was found ( Figure 1H ). The dynamic changes in WBC and eosinophil in this patient during treatment are shown ( Figure 1I ).