DOI: 10.17633/rd.brunel.8016191.v1
title: The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): revised characterisation of cytogenetics features

description: <b>Figure 1. </b><b>Representative karyotype of RS4;11 obtained by M-FISH.</b> 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).<br> <b></b> <b>Figure 2. Cytogenetic abnormalities investigated by FISH in the RS4;11 cell line. </b>(<b>A-B</b>) Whole chromosome paints for chromosomes X and 18 did not reveal any numerical abnormalities. (<b>C-D</b>) 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. (<b>E</b>) Whole chromosome paint for chromosome 8 revealed a size difference between chromosomes 8, with one copy appearing metacentric. (<b>F</b>) A duplication of the 8q24 region is visible on an isochromosome 8q, detected with the FISH probe RP11-195E4 specific for 8q24.3. (<b>G</b>) Dual-colour FISH probe CDKN2A/2B XL showed a homozygous deletion of the 9p21 locus by observation of green centromeric signals only. (<b>H</b>) A representative metaphase from the Farage cell line with two normal chromosomes 9 with the expected pattern for the CDK24A/2B XL probe.<b></b> <br> <b>Figure 3. Dual-colour FISH shows the <i>KMT2A </i>rearrangement in RS4;11. </b>FISH using a break-apart, dual-colour probe mapping proximal (red) and distal (green) to the <i>KMT2A</i> 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).<br> <br> <b><br> </b> <b>Figure 4. The presence of the <i>KMT2A-AFF1</i> transcript in RS4;11 cells confirmed by RT-PCR and Sanger sequencing. </b>(<b>A</b>) Schematic representation of the position of primers flanking the fusion fragment by the forward MLL-C and reverse AF4-D. (<b>B</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 <i>KMT2A</i> transcript: ENST00000534358.5; and for <i>AFF1</i> transcript: ENST00000307808.10. (<b>C</b>) 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). (<b>D, E, F</b>) Representative sequencing chromatograms of the <i>KMT2A-AFF1</i> junction in the PCR product (<b>D</b>) and in two distinct clones (<b>E, F</b>). The nucleotides of <i>KMT2A</i> exon 9 are highlighted in blue. The chromatogram was generated using FinchTV software. (<b>G</b>) Schematic representation of the <i>KMT2A</i> exon 9-intron-<i>AFF1</i> exon 4 boundaries demonstrating how the alternative splicing can generate a canonical <i>KMT2A-AFF1</i> transcript, corresponding to the chromatogram in (<b>E</b>) or the transcript containing the deletion of three nucleotides at the beginning of <i>AFF1</i> exon 4 that corresponds to the chromatogram in (<b>F</b>). <br>