PMC6542082_01
G-banding, FISH, and aCGH analyses. a The G-banding analysis showed a normal karyotype, 46,XX. b Interphase FISH with the PDGFRB breakapart probe on a normal nucleus and on a nucleus with loss of the distal probe suggesting a genetic breakpoint in the PDGFRB locus. c aCGH showing deletions on chromosome bands 5q32q35.3, 7q34, 9p13, 10q26.13, 14q11.2, and 14q32.33
40164_2019_136_Fig1_HTML.jpg
PMC6542082_01_40164_2019_136_Fig1_HTML.jpg
Fig1
The G-banding analysis at diagnosis of bone marrow and blood metaphase cells revealed a normal karyotype, 46,XX, in all 25 examined metaphases (Fig. 1a). FISH analyses with the Cytocell (Cytocell, Banbury, Oxfordshire, UK) multiprobe ALL panel did not detect aberrations of MYC, CDKN2A, TCF3, MLL, and IGH, no ETV6-RUNX1 or BCR-ABL1 fusions, nor was hyperdiploidy seen in 200 examined nuclei (data not shown). FISH with the PDGFRB breakapart probe (Cytocell) showed loss of the distal part of the probe in 176 out of 201 examined interphase nuclei from white blood cells and 42 out of 100 examined interphase nuclei from bone marrow cells, suggesting a genetic breakpoint in the PDGFRB locus in 5q32 (Fig. 1b).  Table 1). The deletion on 5q was 30 Mb long, started between exons 8 and 9 of PDGFRB (5q32), and finished in the CANX locus (5q35.3). The result was in agreement with the FISH data obtained with the PDGFRB breakapart probe (Fig. 1b, c).
Molecular genetic and FISH analyses for identification of the GTF2I-PDGFRB fusion gene. a The 76 bp sequence obtained from the raw data of RNA sequencing using the command "grep". The search term "TCCCTGTCCGAGTGCTGG" is underlined. b Partial sequence chromatogram of the PCR amplified cDNA fragment showing the fusion (arrow) of GTF2I and PDGFRB. c Partial sequence chromatogram of the PCR amplified genomic DNA fragment showing the fusion (arrow) of GTF2I and PDGFRB. The identification of the GTF2I gene as the PDGFRB-fusion partner was possible only after FISH experiments using appropriate commercial BAC probes for PDGFRB and GTF2I. d Ideogram of chromosome 5 showing the mapping position of the PDGFRB gene (vertical red line). e Diagram showing the FISH probe RP11-21I20 for PDGFRB. Additional genes in this region are also shown. f Ideogram of chromosome 7 showing the mapping position of the GTF2I gene (vertical green line). g Diagram showing the FISH probe RP11-137E8 for GTF2I. Additional genes in this region are also shown. h FISH on interphase nuclei with the PDGFRB (red signal) and GTF2I (green signal) probes showing a red signal, a green signal and one yellow-fusion signal (arrow)
40164_2019_136_Fig2_HTML.jpg
PMC6542082_01_40164_2019_136_Fig2_HTML.jpg
Fig2
Using the search term "TCCCTGTCCGAGTGCTGG", which corresponds to 1713-1730 nt in the PDGFRB reference sequence with accession number NM_002609.3, only one 76 bp long sequence was extracted (Fig. 2a). Sanger sequencing of the PCR products verified the fusion which was found upon searching the RNA sequencing data using the "grep" command (Fig. 2b). GTF2IP1-PDGFRB, or GTF2I-PDGFRB, or GTF2IP4-PDGFRB. Genomic PCR with the primers GTF2I-3317F1 and PDGFRB-1737R1 amplified a single 1200 bp fragment which by Sanger sequencing was shown to be a hybrid genomic DNA fragment in which intron 8 of PDGFRB is fused with either intron 22 of GTF2IP1, intron 32 of GTF2I, or intron 22 of GTF2IP4 (Fig. 2c). Additional interphase FISH experiments were performed to detect the GTF2I-PDGFRB fusion gene (Fig. 2d-h). RP11-137E8 were retrieved from the Human "32K" BAC Re-Array library (BACPAC Resources, https://bacpacresources.org/home.htm). RP11-21I20, the probe for the PDGFRB gene, mapped to band 5q32 (Position: chr5: 149,320,375-149,496,703; UCSC Genome Browser on Human February 2009 GRCh37/hg19 Assembly) and was labeled red (Fig. 2d, e). RP11-137E8, the probe for the GTF2I gene, mapped to band 7q11.23 (Position: chr7: 73,944,720-74,129,587) and was labeled green (Fig. 2f, g). Fluorescent signals were captured and analyzed using the CytoVision system (Leica Biosystems, Newcastle, UK). FISH analysis showed a fusion signal in 44 out of 100 examined interphase nuclei from bone marrow cells suggesting a GTF2I-PDGFRB fusion gene (Fig. 2h). Thus, FISH with specific probes for PDGFRB and GTF2I was crucial to show that a novel GTF2I-PDGFRB fusion gene had been formed (Fig. 2d-h). 
Molecular genetic and FISH analyses for identification of the IKZF1-TYW1 fusion gene. a The sequence which was obtained using the FusionCatcher software with the fastq files of the RNA sequencing data. b Partial sequence chromatogram of the PCR amplified cDNA fragment showing the fusion (arrow) of IKZF1 and TYW1. c Ideogram of chromosome 7 showing the mapping position of IKZF1 (vertical red line) and TYW1 (vertical green line). d Diagram showing the FISH probe (RP11-813K3 and RP11-95E2) for IKZF1. Additional genes in this region are also shown. e Diagram showing the FISH probe (RP11-458F8 and RP11-166O4) for TYW1. Additional genes in this region are also shown. f FISH on interphase nuclei with the IKZF1 (red signal) and TYW1 (green signal) probes showing a red signal, a green signal, and two yellow-fusion signals (arrow)
40164_2019_136_Fig3_HTML.jpg
PMC6542082_01_40164_2019_136_Fig3_HTML.jpg
Fig3
RT-PCR with the primers IKZF1-469F1/TYW1-1282R1 (Table 2) amplified a 319 bp long cDNA fragment which by Sanger sequencing was shown to contain IKZF1-TYW1 (Fig. 3b). The fusion point thus detected was identical to that found by analysis of the RNA sequencing data using the FusionCatcher software (Fig. 3a, b). In the IKZF1-TYW1 transcript, exon 4 of IKZF1 (nt 642 in sequence with accession number NM_006060 version 6) was fused out-of-frame to exon 8 of TYW1 (nt 1131 in NM_018264 version 4) (Fig. 3b). The probe for the IKZF1 gene was constructed from a pool of the clones RP11-813K3 (Accesion number AC020743; Position: chr7: 50157413-50339940) and RP11-95E2 (Accesion number AC018705; chr7: 50475184-50648153) and was labeled red (Fig. 3c, d). The probe for the TYW1 gene was constructed from a pool of the clones RP11-458F8 (Accesion number AC073335; Position: chr7: 66297269-66454983) and RP11-166O4 (Accesion number AC006480; Position: Chr7: 66699524-66859231) and was labeled green (Fig. 3c, e).