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Reverse panhandle PCR identifies RIBOSOMAL PROTEIN S3 as a new partner gene of MLL in a three-way MLL rearrangement in infant acute monoblastic leukemia

Reverse panhandle PCR identifies RIBOSOMAL PROTEIN S3 as a new partner gene of MLL in a three-way MLL rearrangement in infant acute monoblastic leukemia

Blood 102(11): 184b-185b, November 16

Most myelomonocytic and monoblastic variants of AML in infants and young children are characterized by chromosomal translocations of the MLL gene at chromosome band 11q23. A small but significant fraction of MLL translocations are complex rearrangements. We performed cytogenetic and molecular characterization of a 3-way MLL rearrangement and discovered the RIBOSOMAL PROTEIN S3 (RPS3) gene from chromosome band 11q13.3-11q13.5 as a new partner gene of MLL in infant monoblastic AML. Recently we developed reverse panhandle PCR for cloning unknown genomic breakpoint junctions of other derivative chromosomes of MLL translocations as a method especially well-suited to 5'-partner gene-MLL-3' breakpoint junctions of complex 3-way rearrangements. Here we used a new adaptation of reverse panhandle PCR to characterize a complex 3-way rearrangement. FAB M5 AML was diagnosed at 4 months of age. The conventional Q-banded karyotype suggested t(10;11)(p12;q13). However, FISH with a probe for MLL showed an ins(10;11)(p12;q23q13), indicating a more complex 3-way rearrangement. Southern blot analysis of the MLL bcr showed apprx6.2 kb and 14.0 kb MLL bcr rearrangements, confirming MLL involvement in this translocation. The stem-loop template for reverse panhandle PCR was created after attaching sequence from 3' in the MLL bcr to the unknown 5' partner sequence, and the intervening sequence was amplified with two primers from 3' in the bcr designed to anneal to either end of the stem-loop template. Reverse panhandle PCR gave a 5882 bp product consistent with the 6.2 kb rearrangement. The reverse panhandle PCR product contained two genomic breakpoint junctions separated by 131-135 bases of an inverted sequence of the genomic clone corresponding to the RPS3 gene (GenBank no. AF281313) at chromosome band 11q13.3-11q13.5, confirming the disruption of band 11q13 by the complex rearrangement. The more 5' breakpoint junction fused position 2004 of RPS3 intron 5 with position 2126 of RPS3 intron 5 sequence in an inverted orientation. The more 3' breakpoint junction fused position 1996, 1995, 1994, 1993 or 1992 of the inverted RPS3 sequence to position 3305, 3306, 3307, 3308 or 3309 in MLL intron 8. Sequence microhomologies between RPS3 and the inverted sequence and between RPS3 and MLL at the respective breakpoint junctions were consistent with DNA repair via the NHEJ. The sequence microhomologies precluded exact breakpoint assignments at the 5'-RPS3-MLL-3' breakpoint junction. The RPS3 DNA segments immediately at both genomic breakpoint junctions contained palindromic sequences of 12-base inverted repeats that were duplicated in the recombination. The reverse panhandle PCR technique proved effective for cloning the unknown 5'-partner gene-MLL-3' breakpoint junction in the 3-way rearrangement. Characterization of each breakpoint junction is important for an understanding of the translocation mechanism in complex translocations. It is conceivable that formation of semistable cruciform loops from the palindromic regions rendered RPS3 vulnerable to translocation. Additional studies are underway to determine if there is a 5'-RPS3-MLL-3' transcript. The RPS3 protein is involved in the initiation of translation and would be a new type of partner protein. The 5'-MLL-partner gene-3' genomic breakpoint junction and resulting fusion transcript will also be examined. The complex rearrangement of chromosomes 10, 11 and 11 fusing RPS3 and MLL is a novel 3-way translocation. RPS3 increases the heterogeneity of the now >40 partner genes disrupted by MLL translocations.

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Accession: 035666030

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