AF5q31, a newly identified AF4-related gene, is fused to MLL in infant acute lymphoblastic leukemia with ins(5;11)(q31;q13q23)

Abstract

Infant acute lymphoblastic leukemia (ALL) with MLL gene rearrangements is characterized by early pre-B phenotype (CD10(-)/CD19(+)) and poor treatment outcome. The t(4;11), creating MLL-AF4 chimeric transcripts, is the predominant 11q23 chromosome translocation in infant ALL and is associated with extremely poor prognosis as compared with other 11q23 translocations. We analyzed an infant early preB ALL with ins(5;11)(q31;q13q23) and identified the AF5q31 gene on chromosome 5q31 as a fusion partner of the MLL gene. The AF5q31 gene, which encoded a protein of 1,163 aa, was located in the vicinity of the cytokine cluster region of chromosome 5q31 and contained at least 16 exons. The AF5q31 gene was expressed in fetal heart, lung, and brain at relatively high levels and fetal liver at a low level, but the expression in these tissues decreased in adults. The AF5q31 protein was homologous to AF4-related proteins, including AF4, LAF4, and FMR2. The AF5q31 and AF4 proteins had three homologous regions, including the transactivation domain of AF4, and the breakpoint of AF5q31 was located within the region homologous to the transactivation domain of AF4. Furthermore, the clinical features of this patient with the MLL-AF5q31 fusion transcript, characterized by the early pre-B phenotype (CD10(-)/CD19(+)) and poor outcome, were similar to those of patients having MLL-AF4 chimeric transcripts. These findings suggest that AF5q31 and AF4 might define a new family particularly involved in the pathogenesis of 11q23-associated-ALL.

Figure 1

Southern blot of DNA digested with HindIII and probed with the 0.9-kb fragment of the MLL gene. C, normal peripheral lymphocytes. P, leukemic cells from the patient. The patient exhibited a rearranged band (arrow) with this probe.

Figure 2

(a) Partial sequence of the junction of the MLL-AF5q31 chimeric transcript. The arrow indicates the fusion point. (b) Cloning of AF5q31 cDNA clones. (c) Sequencing of the AF5q31 cDNA. * indicates a termination codon. The arrow indicates the breakpoint in the patient. (d) Sequence of the ins(5;11)(q31;q13q23) breakpoint region in the patient. (e) Physical map of the chromosome 5q31-cytokine cluster region and P1 clones.

Figure 3

(a) Comparison of predicted coding sequence of AF5q31 with other related sequences. (b) Schematic representation of homology between AF5q31 and AF4 proteins. The percentage of amino acid homology between corresponding regions of AF5q31 and AF4 is indicated. Arrowheads indicate the fusion points with MLL.

Figure 4

Detection of the MLL-AF5q31 chimeric transcripts by reverse transcription–PCR. Primers used were MLL-7S and 5–1A (lane 1), and 5–13S and MLL-11A (lane 2), respectively. M, size marker.

Figure 5

Northern blot analysis of RNAs from adult (a) and fetal (b) human tissues. UAF5q31 and AF4 cDNA fragments were used as probes for the Northern blots in the upper and middle figures, respectively. Membranes were rehybridized to the β-actin probe for the lower figures. The organs from which tissues were analyzed are indicated on top of each lane.

Figure 6

Chromosomal localization of the AF5q31 gene. (a) A fluorescence in situ hybridization pattern obtained with the phage clone λH17–6 as a probe. (b) The same metaphase spread stained with 4′,6-diaminido-2-phenylindole dihydrochloride (DAPI). A DAPI image was inverted and enhanced in terms of band image contrast. The AF5q31 gene was assigned to band 5q31.1 as indicated by arrows.