Figure 1
XPO1-MLLT10 fusion detected by RNA-sequencing is associated with deregulation of HOXA gene locus expression. (A) Expression of HOXA9 in genetic subgroups of T-ALL. Levels were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and calculated relative to an ABL housekeeping gene control. Boxes encompass the 25th through 75th percentiles, with the horizontal bar denoting the median expression level. Whiskers indicate the 10th and 90th percentiles. The numbers of cases tested in each group were as follows: XPO1-MLLT10, n = 1; PICALM-MLLT10, n = 36; SET-NUP214, n = 18; MLL-AF6, n = 9; TLX1+, n = 86; TLX3+, n = 67; SIL-TAL, n = 40. (B) Upper panel: Genomic mapping of the XPO1-MLLT10 fusion by poly(A)-enriched strand-specific RNA-sequencing using the SOLiD HQ5500xl system (Life Technologies). Mapping, coverage, and fusion discovery were determined by using LifeScope (Life Technologies), with reference to version hg19 of the human genome. A schematic representation of paired-end and fusion-spanning reads that revealed fusion between exon 24 of XPO1 (chr2:61708320-61708416) and exon 6 of MLLT10 (chr10:21901277-21901380) is shown. Solid lines indicate split reads spanning 2 exons, and dotted lines indicate 2 reads of the same fragment. The numbers of unique reads for the wild-type XPO1 (exons 24 and 25) and MLLT10 (exons 5 and 6) transcripts are also depicted. Lower panel: Confirmation of expression of an in-frame XPO1-MLLT10 fusion transcript by direct (Sanger) sequencing. The positions of the nucleotide (NT) and amino acid (AA) at the breakpoint of each gene are annotated. (C) Expression of HOXA genes in XPO1-MLLT10+ (n = 1; denoted by triangles) and PICALM-MLLT10+ (n = 4; mean levels denoted by circles with error bars indicating standard error of the mean) blasts. Transcript quantification was determined by qPCR using a TaqMan Low-Density Array, and the results of 2 experimental replicates were combined. Expression was calculated relative to a GAPDH housekeeping gene control. (D) RT-PCR for XPO1-MLLT10; 84 cases of HOXA+ T-ALL lacking known explicatory genetic anomalies were screened by using primers specific for the XPO1-MLLT10 fusion transcript (product size, 618 bp). A representative PCR result is shown. T-ALL 1 is the index XPO1-MLLT10+ case. NTC, no template control; PE, paired-end; SE, single-end.

XPO1-MLLT10 fusion detected by RNA-sequencing is associated with deregulation of HOXA gene locus expression. (A) Expression of HOXA9 in genetic subgroups of T-ALL. Levels were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and calculated relative to an ABL housekeeping gene control. Boxes encompass the 25th through 75th percentiles, with the horizontal bar denoting the median expression level. Whiskers indicate the 10th and 90th percentiles. The numbers of cases tested in each group were as follows: XPO1-MLLT10, n = 1; PICALM-MLLT10, n = 36; SET-NUP214, n = 18; MLL-AF6, n = 9; TLX1+, n = 86; TLX3+, n = 67; SIL-TAL, n = 40. (B) Upper panel: Genomic mapping of the XPO1-MLLT10 fusion by poly(A)-enriched strand-specific RNA-sequencing using the SOLiD HQ5500xl system (Life Technologies). Mapping, coverage, and fusion discovery were determined by using LifeScope (Life Technologies), with reference to version hg19 of the human genome. A schematic representation of paired-end and fusion-spanning reads that revealed fusion between exon 24 of XPO1 (chr2:61708320-61708416) and exon 6 of MLLT10 (chr10:21901277-21901380) is shown. Solid lines indicate split reads spanning 2 exons, and dotted lines indicate 2 reads of the same fragment. The numbers of unique reads for the wild-type XPO1 (exons 24 and 25) and MLLT10 (exons 5 and 6) transcripts are also depicted. Lower panel: Confirmation of expression of an in-frame XPO1-MLLT10 fusion transcript by direct (Sanger) sequencing. The positions of the nucleotide (NT) and amino acid (AA) at the breakpoint of each gene are annotated. (C) Expression of HOXA genes in XPO1-MLLT10+ (n = 1; denoted by triangles) and PICALM-MLLT10+ (n = 4; mean levels denoted by circles with error bars indicating standard error of the mean) blasts. Transcript quantification was determined by qPCR using a TaqMan Low-Density Array, and the results of 2 experimental replicates were combined. Expression was calculated relative to a GAPDH housekeeping gene control. (D) RT-PCR for XPO1-MLLT10; 84 cases of HOXA+ T-ALL lacking known explicatory genetic anomalies were screened by using primers specific for the XPO1-MLLT10 fusion transcript (product size, 618 bp). A representative PCR result is shown. T-ALL 1 is the index XPO1-MLLT10+ case. NTC, no template control; PE, paired-end; SE, single-end.

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