1. Identification of Multiple Complex Rearrangements Associated with Deletions in the 6q23-27 Region in Sézary Syndrome 
Katarzyna Iżykowska, Mariola Zawada, Karina Nowicka, Piotr Grabarczyk, Floriane C.M. Braun, Martin Delin, Markus Möbs, Marc Beyer, Wolfram Sterry, Christian A. Schmidt, Grzegorz K. Przybylski 
Journal of Investigative Dermatology 
Volume 133, Issue 11, Pages (November 2013)
DOI: /jid
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2. Figure 1
Complex rearrangements identified by combining fine-tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR). Three different rearrangements, transposition, deletion, and inversion, all involving a duplicated 1,064-kb region, were identified in Sézary syndrome (SS) patient 247. In the picture, the inversion identified by LM-PCR is presented (rearrangement nr 14, amplified with primers 148,702,669f and 148,702,771f). This inversion and the deletion disrupted the MYB gene, localized at the distal end of the duplicated region. C, control sample; G, germ-line configuration; M, size marker; R, rearrangement.
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
Combination of fine-tiling comparative genomic hybridization (FT-CGH) and paired-end next-generation sequencing (NGS) for identification of rearrangements in the 6q23-27 region in Sézary syndrome (SS) patient 312. Region 150.5–150.8M was enlarged to indicate the inverted 164,252-bp DNA fragment and gene fusions that arose as a result of this rearrangement. IYD, iodotyrosine deiodinase gene.
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4. Figure 3
6q23-27 rearrangements in Sézary syndrome cell line (SeAx) cell line identified by fine-tiling comparative genomic hybridization (FT-CGH) and next-generation sequencing. (a) FT-CGH analysis showed deletions in the 6q23-27 region in SeAx; breakpoints were characterized using ligation-mediated PCR (LM-PCR), and five rearrangements were identified; all rearrangements were confirmed using paired-end sequencing, and one breakpoint was identified to be associated with telomere repeats; two gene fusions were identified at the DNA level, yet only IL22RA2/CCDC28A fusion had the orientation to create a possible transcript. (b) Two inversions in SeAx cell line identified by FT-CGH/LM-PCR and paired-end sequencing; biallelic deletions visualized with both techniques.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Translocation t(6;17) in SeAx fusing the AIG1 and the GOSR1 genes. Discordant next-generation sequencing (NGS) reads paired with DNA fragments from chromosomes 3 and 17 are indicated by circles.
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6. Figure 5
Cytogenetic confirmation of the rearrangements identified by next-generation sequencing Sézary syndrome (SeAx) cell line. (a) Fluorescence in situ hybridization (FISH) analysis of the SeAx cell line with whole chromosome painting probes for chromosome 6 (green) and chromosome 3 (red); chromosome aberrations recognized: der(3)t(3;?), der(?)t(?;3), der(3)t(3;6;?), der(3)t(3;6), der(6)t(6;?), der(?)t(?;6), del(6)(q?). (b) Multicolor FISH (mFISH) analysis of the whole SeAx cell line karyotype; detailed analysis was concentrated on chromosomes 6 and 3; G-banding was performed in order to assess the band designation: der(3)t(3;6;17;9)(3pter→3q13.2::6q22→6q24::17q11→17q23?::9q22.3→9qter); der(3)t(3;6)(3p26→3q13?::6q25?→6q27?); der(17)t(3;17)(3qter→3q26::17q11→17p12); der(17)t(3;17)(17qter→17p11?::3q21→3qter). (c) Reconstruction of the sandwich chromosome der(3)t(3;6;17;9) detected in mFISH based on molecular results; the presence of the 5,797-bp insert was detected by next-generation sequencing (NGS) but not by mFISH, and it was confirmed on genomic DNA using one primer situated in the 6q21 region (position 106,549,156r) and the second one in the 3q13.2 region (position 113,775,810f).
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7. Figure 6
Quantitative expression analysis of genes disrupted by a rearrangement. Expression of three genes (MYB, PERP, and IL22RA2) directly affected by a rearrangement and three genes (MYC, HSPA8, and BCL2) regulated by MYB was analyzed using real-time quantitative PCR with specific TaqMan probes in six Sézary syndrome (SS) patients with and seven SS patients without 6q23-27 alterations, two SS cell lines (SeAx and HuT-78), and five mononuclear cell samples (MNC) from healthy donors. Each population, as well as the patients with a particular gene rearrangement, is featured by different colors. The gene copy number was normalized to 1 million copies of beta 2 microglobulin (B2MG) reference gene. In the MYC expression diagram, information on MYC amplification (M) and E2A deletions (E) in 8/13 SS patients, published elsewhere by Steininger et al., 2011, positively contributing to MYC expression has been included. No alterations: (–). In addition, TP53 deletions (T) have been indicated, which might suppress the MYC apoptotic pathway. The level of gene expression in cell lines that were out of range was given by numbers.
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