Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR-154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer and suggests a role in the aetiology of leukaemia.

We have developed a directly quantitative method utilizing genomic clone DNA microarrays to assess the replication timing of sequences during the S phase of the cell cycle. The genomic resolution of the replication timing measurements is limited only by the genomic clone size and density. We demonstrate the power of this approach by constructing a genome-wide map of replication timing in human lymphoblastoid cells using an array with clones spaced at 1 Mb intervals and a high-resolution replication timing map of 22q with an array utilizing overlapping sequencing tile path clones. We show a positive correlation, both genome-wide and at a high resolution, between replication timing and a range of genome parameters including GC content, gene density and transcriptional activity.

Genome-wide single nucleotide polymorphism analysis has revealed large-scale cryptic regions of acquired homozygosity in the form of segmental uniparental disomy in f20% of acute myeloid leukemias. We have investigated whether such regions, which are the consequence of mitotic recombination, contain homozygous mutations in genes known to be mutational targets in leukemia. In 7 of 13 cases with uniparental disomy, we identified concurrent homozygous mutations at four distinct loci (WT1, FLT3, CEBPA, and RUNX1). This implies that mutation precedes mitotic recombination which acts as a ''second hit'' responsible for removal of the remaining wildtype allele, as has recently been shown for the JAK2 gene in myeloproliferative disorders.

MicroRNAs (miRNAs) are short single-stranded RNAs that have a potentially important role in gene regulation. Using a quantitative real-time polymerase chain reaction assay specific to the mature miRNA, the expression level of a selected group of haematopoietic tissue-specific miRNAs was measured across a set of 30 primary adult acute myeloid leukaemia (AML) with a normal karyotype. The expression levels of each miRNA were correlated with the genome-wide mRNA expression profiles in the same leukaemias. This revealed that miR181a correlated strongly with the AML morphological sub-type and with the expression of genes previously identified through sequence analysis as potential interaction targets. Three other miRNAs, miR-10a, miR-10b and miR-196a-1, showed a clear correlation with HOX gene expression. Leukemia (2007) 21, 912-916.

Gene expression profiles were determined from presentation peripheral blood and bone marrow samples of 28 patients with acute myeloid leukemia (AML). Hierarchical clustering sorted the profiles into separate groups, each representing one of the major cytogenetic classes in AML [i.e., t(8;21), t(15;17), inv(16), 11q23, and normal karyotype]. Statistical group comparison identified genes whose expression was strongly correlated with these chromosomal classes. Moreover, the normal karyotype AMLs were characterized by distinctive up-regulation of certain members of the class I homeobox A and B gene families, implying a common underlying genetic lesion. These data reveal novel diagnostic and therapeutic targets and demonstrate the potential of microarray-based dissection of AML.

NOTESupplementary Figure 1S can be found at http:// www.landesbioscience.com/journals/cc/woodfine CC4-1-sup.pdf. ReportReplication Timing of Human Chromosome 6 ABSTRACT Genomic microarrays have been used to assess DNA replication timing in a variety of eukaryotic organisms. 1-3 A replication timing map of the human genome has already been published at a 1Mb resolution. 3 Here we describe how the same method can be used to assess the replication timing of chromosome 6 with a greater resolution using an array of overlapping tile path clones. We report the replication timing map of the whole of chromosome 6 in general, and the MHC region in particular. Positive correlations are observed between replication timing and a number of genomic features including GC content, repeat content and transcriptional activity.

Many cases of AML have either a normal karyotype or non-recurrent chromosomal abnormalities and hence their pathogenesis remains obscure. The introduction of array-based analysis of single nucleotide polymorphisms (SNPs) allows the rapid determination of genome-wide allelic information at a high density for a DNA sample. High-resolution SNP genotype analysis was performed on 64 presentation AML samples with full karyotype information as follows: normal karyotype [40], t(8;21) [5], t(15;17) [4], inv16 [3], 11q23 [2],−7 [3],+8 [2] and other structural abnormalities [7]. Using the 10K SNP array (Affymetrix, Inc., Santa Clara) 9, a mean call rate of 93.3% yielded more than 10,000 SNP genotype calls per sample. Large unexpected regions of homozygosity were observed in 12 AMLs (18.75%). These regions ranged in size from 16 million base pairs to 113 million base pairs and would have been visible in the karyotypes if due to deletion. Remission bone marrow samples from 5 of those patients were subjected to SNP genotype analysis. The SNP call data demonstrated clearly that the homozygosity seen in the leukemic DNA was not present in the respective remission bone marrow DNA. Fluorescence in situ hybridisation (FISH) demonstrated 2 signals for probes within regions of homozygosity. Furthermore, hybridisation signal values on the SNP arrays demonstrated that regions of homozygosity did not differ from the rest of the chromosome. It was therefore concluded that such homozygous regions corresponded to uniparental disomy (UPD) due to somatic recombination events occurring during development of the leukemias. There appears to be a non-random distribution of UPD with 5 events on chromosome 11, 2 on chromosome 6, 2 on chromosome 9 and 1 on chromosomes 13, 19 and 21. As expected for somatic recombination, homozygosity continued to the telomere in most cases. Any parental bias in UPD could be evidence of a role for imprinted genes. This issue was investigated using the H19 gene, which is located at 11p15 and is normally methylated only on the paternal allele. Two leukemias exhibited UPD including 11p15 and the methylation status of the H19 gene was therefore determined by bisulfite sequencing. One leukemia with UPD11p exhibited a homozygous methylated paternal pattern, while the other example of UPD11p showed a homozygous non-methylated maternal pattern. These data show that the UPD seen on 11p is not restricted to a single parental origin. In a previous analysis, the leukemia with UPD19q was shown to be homozygous for a CEBPA mutation and FISH demonstrated 2 copies of the CEBPA gene. This gene is located at 19q13.1, within the area of UPD and we conclude that the mutation occurred prior to the UPD. We can therefore speculate that an important consequence of UPD could be to unmask pre-existing mutations. A total of 8 different chromosomal regions have been shown to be affected by UPD in this study and this may suggest that there are at least this number of mutational targets. The discovery of widespread, somatically acquired, UPD in leukemias has potentially important clinical implications. 20% of the normal karyotype AMLs was found to have UPD, and this could offer a valuable new approach to the classification of this important subgroup of AML. The prognostic consequences of such cryptic abnormalities for the patient are uncertain, and larger studies will be required to assess the clinical significance of this phenomenon.

The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.