10.17633/rd.brunel.8016191.v1
Denise ragusa
Denise
ragusa
Evgeny Makarov
Evgeny
Makarov
Oliver Britten
Oliver
Britten
Daniela Moralli
Daniela
Moralli
catherine Green
catherine
Green
Sabrina Tosi
Sabrina
Tosi
The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): revised characterisation of cytogenetics features
Brunel University London
2019
Leukaemia
Leukaemia cell line
RS4;11
Fluorescence in situ Hybridisation
M-FISH
karyotype
RT-PCR
fusion transcript
MLL gene
MLL-AF4
Genetics not elsewhere classified
Hematology
Cancer
2019-06-03 09:27:15
Dataset
https://brunel.figshare.com/articles/dataset/The_RS4_11_cell_line_as_a_model_for_leukaemia_with_t_4_11_q21_q23_revised_characterisation_of_cytogenetics_features/8016191
<div>
<p><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></p>
<p><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></p>
<br>
<p> </p>
<p><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>
</p>
<br>
<p> </p>
<b><br>
</b>
<p> </p>
<p><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>).</p>
<br></div>