Molecular characterization of MLL-FRYL translocation. Marrow from 19 months after neuroblastoma diagnosis, which was 82% replaced by cells with t(4;11)(p12;q23) by FISH and 90% replaced by cells with t(4;11)(p12;q23) by karyotype analysis, was used for Southern blot and molecular cloning. (A) Southern blot analysis performed with B859 cDNA probe²⁷  and indicated restriction enzyme digests. Dashes indicate germline bands; arrows show rearrangements. (B) BglII panhandle PCR gave a 6.0-kb BglII germline product from normal MLL allele but no larger or smaller product consistent with a rearrangement (left). BglII reverse-panhandle PCR product, the size of which indicated that 3.2-kb BglII fragment on the Southern blot (panel A, circled) was der(4) rearrangement (right). Vertical lines have been inserted to indicate repositioned gel lanes. (C) Summary of der(4) genomic breakpoint junction in subclones of BglII reverse panhandle PCR product generated by TOPO TA cloning (Invitrogen). One subclone was sequenced in entirety; the breakpoint junction was verified in 2 more subclones and confirmed by PCR with gene-specific primers. Fifty-nine base pairs of 5′ sequence are from MLL primer 3 through ligated oligonucleotide (P-oligo); 418-421 bp of 5′ sequence are the partner gene. The partner DNA sequence corresponded with GenBank accession no. NM_015030 (FRYL, KIAA0826). The 3′ 2512-2515 bp include MLL bcr sequence beginning at position 5486, 5487, 5488, or 5489 (GenBank accession no. U04737), which is in intron 8(11) through MLL primer 4. Brackets show possible FRYL and MLL breakpoint positions and 5′-AGT-3′ in both genes that precluded more precise breakpoint assignments (bottom). Repetitive sequences are shown (middle). MLL intron number is Rasio et al²⁴  designation with parenthetical Nilson et al²⁵  designation alongside. (D) PCR product obtained with gene-specific primers containing der(11) genomic breakpoint junction, which was directly sequenced. MLL and FRYL breakpoints (brackets) could not be assigned precisely because of 5′-AA-3′ in both genes. Short sequence homologies (brackets) and loss of 8-13 bases from MLL and 31-36 bases from FRYL, indicated by comparison with the der(4) genomic breakpoint junction (B), suggested imprecise NHEJ repair.