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Identification of Mixed Lineage Leukemia Gene (MLL)/MLLT10 Fusion Transcripts by Reverse Transcription-PCR and Sequencing in a Case of AML With a FISH-Negative Cryptic MLL Rearrangement.

Ko K, Kwon MJ, Woo HY, Park H, Park CH, Lee ST, Kim SH - Ann Lab Med (2015)

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea.

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RT-PCR analysis was performed with a combination of 5' MLL and 3' MLLT10 primers: MLL Forward 5'-GGAAGTCAAG CAAGCAGGTC-3' and MLLT10 Reverse 5'-GCTGCCATTGATGAATTTG-3'... RT-PCR revealed a 386-bp product ranging from the exon 8 region of the MLL gene to the exon 16 region of the MLLT10 gene, linked via two orphan nucleotides (Fig. 3)... Currently, long-distance inverse PCR (LDI-PCR) is the most powerful, verified method for detecting known and unknown partner genes in MLL rearrangements... In our case, a genetic aberration, an MLL (exon 8)-MLLT10 (exon 16) rearrangement, was confirmed by sequencing... However, we were not able to perform additional testing such as chromosome microarrays or re-testing with different FISH probes (i.e., from a different manufacturer) or LDI-PCR because of insufficient specimens... The MLLT10/AF10 gene was found to be involved in "chromosomal fragment insertions," a complex mechanism that occurs when either a fragment of chromosome 11 (including portions of the MLL gene) is inserted into another chromosome, or a fragment of another chromosome (including portions of a translocation partner gene) is inserted into the breakpoint cluster region of the MLL gene, and is most frequently involved in the latter mechanism... There is a high likelihood that our case also involved a complex rearrangement, considering that the 3' portion of the MLL gene was fused to the 5' portion of the MLLT10 gene... To the best of our knowledge, this is the first case report of a cryptic MLL rearrangement detected by RT-PCR only, despite testing negative for FISH... However, this case may not represent a truly cryptic MLL rearrangement, considering that the chromosome results revealed a complex karyotype... While FISH assays are recognized for their usefulness in detecting cryptic MLL rearrangements, this case report emphasizes that RT-PCR can be useful for detecting FISH-negative cryptic MLL rearrangements... It would be useful to document other similar cases so that a strategy for diagnosis can be developed.

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Related in: MedlinePlus

Identification of the MLL/MLLT10 fusion transcript in acute myeloid leukemia. Sequencing analyses confirmed that the transcript was from MLL (exon 8)-MLLT10 (exon 16) rearrangement.Abbreviation: MLL, mixed lineage leukemia gene.
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Figure 3: Identification of the MLL/MLLT10 fusion transcript in acute myeloid leukemia. Sequencing analyses confirmed that the transcript was from MLL (exon 8)-MLLT10 (exon 16) rearrangement.Abbreviation: MLL, mixed lineage leukemia gene.

Mentions: Chromosomes were analyzed via GTG banding. Complex structural abnormalities were observed, and the karyotype was 46,XY,der(10)t(10;21)(p12;q21),der(12)del(12)(p11.2)add(12)(q24.2)[17]/46,XY[3] (Fig. 1). FISH analysis was performed according to the manufacturer's instructions, including MLL (LSI Dual-Color, Break Apart Rearrangement Probe, Vysis, Downers Grove, IL, USA). No break-apart signals were observed for the MLL probe (Fig. 2). Multiplex RT-PCR was performed with HemaVision (DNA Technology, Aarhus, Denmark), according to the manufacturer's instructions. Molecular analysis revealed an amplicon corresponding to the MLL/MLLT10 fusion transcript. RT-PCR analysis was performed with a combination of 5' MLL and 3' MLLT10 primers: MLL Forward 5'-GGAAGTCAAG CAAGCAGGTC-3' and MLLT10 Reverse 5'-GCTGCCATTGATGAATTTG-3'. PCR products were subjected to direct sequencing by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). RT-PCR revealed a 386-bp product ranging from the exon 8 region of the MLL gene to the exon 16 region of the MLLT10 gene, linked via two orphan nucleotides (Fig. 3).


Identification of Mixed Lineage Leukemia Gene (MLL)/MLLT10 Fusion Transcripts by Reverse Transcription-PCR and Sequencing in a Case of AML With a FISH-Negative Cryptic MLL Rearrangement.

Ko K, Kwon MJ, Woo HY, Park H, Park CH, Lee ST, Kim SH - Ann Lab Med (2015)

Identification of the MLL/MLLT10 fusion transcript in acute myeloid leukemia. Sequencing analyses confirmed that the transcript was from MLL (exon 8)-MLLT10 (exon 16) rearrangement.Abbreviation: MLL, mixed lineage leukemia gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446590&req=5

Figure 3: Identification of the MLL/MLLT10 fusion transcript in acute myeloid leukemia. Sequencing analyses confirmed that the transcript was from MLL (exon 8)-MLLT10 (exon 16) rearrangement.Abbreviation: MLL, mixed lineage leukemia gene.
Mentions: Chromosomes were analyzed via GTG banding. Complex structural abnormalities were observed, and the karyotype was 46,XY,der(10)t(10;21)(p12;q21),der(12)del(12)(p11.2)add(12)(q24.2)[17]/46,XY[3] (Fig. 1). FISH analysis was performed according to the manufacturer's instructions, including MLL (LSI Dual-Color, Break Apart Rearrangement Probe, Vysis, Downers Grove, IL, USA). No break-apart signals were observed for the MLL probe (Fig. 2). Multiplex RT-PCR was performed with HemaVision (DNA Technology, Aarhus, Denmark), according to the manufacturer's instructions. Molecular analysis revealed an amplicon corresponding to the MLL/MLLT10 fusion transcript. RT-PCR analysis was performed with a combination of 5' MLL and 3' MLLT10 primers: MLL Forward 5'-GGAAGTCAAG CAAGCAGGTC-3' and MLLT10 Reverse 5'-GCTGCCATTGATGAATTTG-3'. PCR products were subjected to direct sequencing by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). RT-PCR revealed a 386-bp product ranging from the exon 8 region of the MLL gene to the exon 16 region of the MLLT10 gene, linked via two orphan nucleotides (Fig. 3).

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

RT-PCR analysis was performed with a combination of 5' MLL and 3' MLLT10 primers: MLL Forward 5'-GGAAGTCAAG CAAGCAGGTC-3' and MLLT10 Reverse 5'-GCTGCCATTGATGAATTTG-3'... RT-PCR revealed a 386-bp product ranging from the exon 8 region of the MLL gene to the exon 16 region of the MLLT10 gene, linked via two orphan nucleotides (Fig. 3)... Currently, long-distance inverse PCR (LDI-PCR) is the most powerful, verified method for detecting known and unknown partner genes in MLL rearrangements... In our case, a genetic aberration, an MLL (exon 8)-MLLT10 (exon 16) rearrangement, was confirmed by sequencing... However, we were not able to perform additional testing such as chromosome microarrays or re-testing with different FISH probes (i.e., from a different manufacturer) or LDI-PCR because of insufficient specimens... The MLLT10/AF10 gene was found to be involved in "chromosomal fragment insertions," a complex mechanism that occurs when either a fragment of chromosome 11 (including portions of the MLL gene) is inserted into another chromosome, or a fragment of another chromosome (including portions of a translocation partner gene) is inserted into the breakpoint cluster region of the MLL gene, and is most frequently involved in the latter mechanism... There is a high likelihood that our case also involved a complex rearrangement, considering that the 3' portion of the MLL gene was fused to the 5' portion of the MLLT10 gene... To the best of our knowledge, this is the first case report of a cryptic MLL rearrangement detected by RT-PCR only, despite testing negative for FISH... However, this case may not represent a truly cryptic MLL rearrangement, considering that the chromosome results revealed a complex karyotype... While FISH assays are recognized for their usefulness in detecting cryptic MLL rearrangements, this case report emphasizes that RT-PCR can be useful for detecting FISH-negative cryptic MLL rearrangements... It would be useful to document other similar cases so that a strategy for diagnosis can be developed.

No MeSH data available.


Related in: MedlinePlus