Diagnostic tool for the identification of MLL rearrangements including unknown partner genes

Abstract

Approximately 50 different chromosomal translocations of the human MLL gene are currently known and associated with high-risk acute leukemia. The large number of different MLL translocation partner genes makes a precise diagnosis a demanding task. After their cytogenetic identification, only the most common MLL translocations are investigated by RT-PCR analyses, whereas infrequent or unknown MLL translocations are excluded from further analyses. Therefore, we aimed at establishing a method that enables the detection of any MLL rearrangement by using genomic DNA isolated from patient biopsy material. This goal was achieved by establishing a universal longdistance inverse-PCR approach that allows the identification of any kind of MLL rearrangement if located within the breakpoint cluster region. This method was applied to biopsy material derived from 40 leukemia patients known to carry MLL abnormalities. Thirty-six patients carried known MLL fusions (34 with der(11) and 2 with reciprocal alleles), whereas 3 patients were found to carry novel MLL fusions to ACACA, SELB, and SMAP1, respectively. One patient carried a genomic fusion between MLL and TIRAP, resulting from an interstitial deletion. Because of this interstitial deletion, portions of the MLL and TIRAP genes were deleted, together with 123 genes located within the 13-Mbp interval between both chromosomal loci. Therefore, this previously undescribed diagnostic tool has been proven successful for analyzing any MLL rearrangement including previously unrecognized partner genes. Furthermore, the determined patientspecific fusion sequences are useful for minimal residual disease monitoring of MLL associated acute leukemias.acute leukemia ͉ MLL translocations ͉ translocation partner genes C hromosomal translocations involving the human MLL gene are recurrently associated with high-risk acute leukemias (1-4). MLL translocations correlate with specific disease subtypes (acute myeloid and acute lymphocytic leukemias), a specific gene expression profile (5, 6), and outcome (favorable or poor), depending on the particular MLL fusion (7). Approximately 50 different MLL translocation partner genes have been identified, suggesting that the human MLL gene is a hot spot for illegitimate recombination events. During illegitimate recombination events, one MLL allele is reciprocally fused with one of the many translocation partner genes. The latter encode nuclear or cytosolic proteins that share only a little sequence homology; however, the fused portion of partner protein sequences is necessary to confer oncogenic potential.The unambiguous identification of these MLL translocations is necessary to support rapid clinical decisions and specific therapy regimens. Current procedures to diagnose MLL rearrangements include cytogenetic analysis, FISH experiments (e.g., split-signal FISH) (8), and specific RT-PCR methods. However, results of RT-PCR analyses are influenced strongly by the quality of the investigated RNA samples. Furthermore, only the most frequent MLL fusions routine...

Backtracking to birth of the NUP98-HOXD13 gene fusion in an infant acute myeloid leukemia

“…After cloning the genomic PCR amplimer deriving from the diagnostic sample and subsequent DNA sequencing, 6 the genomic loci of the chromosomal breakpoint ( Figure 1e) were identified at nucleotide 8850 of NUP98 intron 12 and 3583 nucleotides upstream of HOXD13, thus validating the prior reverse transcriptase-PCR analysis (fusion of NUP98 exon 12 with HOXD13 exon 2). (f) Sequencing analysis of PCR amplimers generated from diagnosis and Guthrie card samples.…”

“…Based on the yet available information, the ND13 in infants correlates with the phenotype AML-M4 and is associated with a good prognosis. By applying state-of-the-art technologies, 6 the genomic breakpoint sequence was unveiled. This allowed us to screen an archived neonatal blood spot to demonstrate for the first time the presence of a concordant ND13 gene fusion.…”

New insights to the MLL recombinome of acute leukemias

“…On the basis of the results obtained in the present (n ¼ 346) and previous studies (414 patients were already published in 2005 and 2006), 11,12 64 translocation partner genes (TPGs) and their specific breakpoint regions have now been identified. Additional 35 chromosomal translocations of the human MLL gene were characterized by cytogenetics, however, without any further molecular characterization.…”

“…11,12 Briefly, 1 mg genomic patient DNA was digested with restriction enzymes and re-ligated to form DNA circles before LDI-PCR analyses. Restriction polymorphic PCR amplimers were isolated from the gel and subjected to DNA sequence analyses to obtain the patient-specific fusion sequences.…”

“…9,10 This repertoire of technologies was recently extended by a long-distance inverse PCR (LDI-PCR) method that uses small amounts of genomic DNA to determine any type of MLL gene rearrangement on the molecular level. 11 This includes chromosomal translocations, complex chromosomal rearrangements, gene internal duplications, deletions or inversions on chromosome 11q and MLL gene insertions into other chromosomes, or vice versa, the insertion of chromatin material into the MLL gene.…”