The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): revised characterisation of cytogenetics features

Figure 1. Representative karyotype of RS4;11 obtained by M-FISH. In this metaphase the karyotype was determined to be 46,XX,t(4;11)(q21;q23),i(7)(q10),i(8)(q10). Arrows indicate the derivatives der(4) and der(11), i(7q) and i(8q).

Figure 2. Cytogenetic abnormalities investigated by FISH in the RS4;11 cell line. (A-B) Whole chromosome paints for chromosomes X and 18 did not reveal any numerical abnormalities. (C-D) An isochromosome 7q was detected using an arm-specific probe (long arm in red and short arm in green), panel C, and a single-locus probe for 7q22 (red) and 7q36 (green), panel D. (E) Whole chromosome paint for chromosome 8 revealed a size difference between chromosomes 8, with one copy appearing metacentric. (F) A duplication of the 8q24 region is visible on an isochromosome 8q, detected with the FISH probe RP11-195E4 specific for 8q24.3. (G) Dual-colour FISH probe CDKN2A/2B XL showed a homozygous deletion of the 9p21 locus by observation of green centromeric signals only. (H) A representative metaphase from the Farage cell line with two normal chromosomes 9 with the expected pattern for the CDK24A/2B XL probe.


Figure 3. Dual-colour FISH shows the KMT2A rearrangement in RS4;11. FISH using a break-apart, dual-colour probe mapping proximal (red) and distal (green) to the KMT2A breakpoint region (A) shows the presence of green signals on the der(4), red signals on the der(11) and a yellow fusion signal on the normal chromosome 11 on metaphase chromosomes (B) and in an interphase nucleus (C).



Figure 4. The presence of the KMT2A-AFF1 transcript in RS4;11 cells confirmed by RT-PCR and Sanger sequencing. (A) Schematic representation of the position of primers flanking the fusion fragment by the forward MLL-C and reverse AF4-D. (B) The predicted sequence of the RT-PCR product amplified by the MLL-C and AF4-D primers (shown in red text and arrows) was estimated to be 502 bp in size. Accession numbers in Ensembl for the KMT2A transcript: ENST00000534358.5; and for AFF1 transcript: ENST00000307808.10. (C) Agarose gel electrophoresis of the amplified fusion product (lane 2), alongside with the non-template control (lane 3). Molecular weight markers from the Bioline HyperLadder I are shown in bp (lane 1). (D, E, F) Representative sequencing chromatograms of the KMT2A-AFF1 junction in the PCR product (D) and in two distinct clones (E, F). The nucleotides of KMT2A exon 9 are highlighted in blue. The chromatogram was generated using FinchTV software. (G) Schematic representation of the KMT2A exon 9-intron-AFF1 exon 4 boundaries demonstrating how the alternative splicing can generate a canonical KMT2A-AFF1 transcript, corresponding to the chromatogram in (E) or the transcript containing the deletion of three nucleotides at the beginning of AFF1 exon 4 that corresponds to the chromatogram in (F).