Cytogenetic and molecular analysis of PAX5-KIAA1549L.
a Interphase FISH using PAX5 exon-specific cosmid probes cos-hPAX5-1 (exons 2–5, red signals) and cos-hPAX5-3 (exons 9–10, green signals) showing loss of a PAX5 3’-end. b RT-PCR using primers specific for PAX5 exon 5 and KIAA1549L exon 20 resulting in amplification of PAX5-KIAA1549L fusion transcripts. Sequencing of the second smaller band, indicated by an asterisk (*), revealed a splice variant of PAX5-KIAA1549L, lacking exon 16 of KIAA1549L. M, molecular weight marker DNA ladder-mix (Peqlab); lane 1, patient; lane 2, normal control; lane 3, water control. c Sequence analysis of the predominant RT-PCR product showing the presence of an in-frame fusion between PAX5 exon 6 and KIAA1549L exon 16. d Schematic representation of the PAX5 wild-type and the PAX5-KIAA1549L proteins. PD, paired domain; 8, octapeptide; HD, partial homeodomain; TA, transactivation domain; ID, inhibitory domain; arrows indicate the nuclear localization signal of PAX5; aa, amino acids. e Chromosomes 9 and 11 with areas of genetic loss (red) detected by the CytoScan HD array and selected genes included in these areas. f Detailed view of PAX5 on chromosome 9 (left panel) and KIAA1549L on chromosome 11 (right panel). The blue dashed lines indicate the breakpoints and the transitions from areas with normal and deleted (red bars) copy number states. The retained exons 1–6 of PAX5 and exons 16–20 of KIAA1549L correspond to the fusion gene detected by RT-PCR