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).

图1. RS4;11的代表性核型图，通过M-FISH获得。在此中期相中，核型被确定为46,XX,t(4;11)(q21;q23),i(7)(q10),i(8)(q10)。箭头指示衍生染色体der(4)和der(11)，i(7q)和i(8q)。

图2. 通过FISH在RS4;11细胞系中研究的染色体异常。（A-B）X染色体和18号染色体的全染色体涂染未发现任何数量异常。（C-D）使用臂特异性探针检测到7q等臂染色体（长臂为红色，短臂为绿色），图C，以及7q22的单位点探针（红色）和7q36（绿色），图D。（E）8号染色体的全染色体涂染显示了8号染色体之间的尺寸差异，其中一份拷贝呈现为中着丝粒。（F）在8q24区域的可见重复，通过针对8q24.3的FISH探针RP11-195E4检测到等臂染色体8q。（G）双色FISH探针CDKN2A/2B XL通过仅观察绿色着丝粒信号显示了9p21位点的纯合子缺失。（H）Farage细胞系的一个代表性中期相，其中两个正常的9号染色体具有CDK24A/2B XL探针预期的模式。

图3. 双色FISH显示了RS4;11中的KMT2A重排。使用断裂分离、双色探针映射KMT2A断裂区域的近端（红色）和远端（绿色）（A）显示中期染色体上der(4)存在绿色信号，der(11)存在红色信号，以及正常染色体11上的黄色融合信号（B）和在间期核（C）中的存在。

图4. 通过RT-PCR和Sanger测序确认了RS4;11细胞中的KMT2A-AFF1转录本的存在。（A）由MLL-C和AF4-D引物环绕融合片段的引物位置示意图。（B）由MLL-C和AF4-D引物扩增的RT-PCR产物的预测序列（以红色文本和箭头显示）估计大小为502碱基。Ensembl中的KMT2A转录本访问号：ENST00000534358.5；和AFF1转录本访问号：ENST00000307808.10。（C）扩增的融合产物的琼脂糖凝胶电泳（泳道2），以及非模板控制（泳道3）。Bioline HyperLadder I的分子量标记以bp表示（泳道1）。（D, E, F）PCR产物中KMT2A-AFF1接头在代表性测序色谱图（D）和两个不同的克隆（E, F）中的存在。KMT2A外显子9的核苷酸以蓝色突出显示。色谱图是用FinchTV软件生成的。（G）KMT2A外显子9-内含子-AFF1外显子4边界的示意图，展示了选择性剪接如何生成经典的KMT2A-AFF1转录本，对应于（E）中的色谱图或包含AFF1外显子4起始处三个核苷酸缺失的转录本，对应于（F）中的色谱图。