RASA4 undergoes DNA hypermethylation in resistant juvenile myelomonocytic leukemia.

Aberrant DNA methylation at specific genetic loci is a key molecular feature of juvenile myelomonocytic leukemia (JMML) with poor prognosis. Using quantitative high-resolution mass spectrometry, we identified RASA4 isoform 2, which maps to chromosome 7 and encodes a member of the GAP1 family of GTPase-activating proteins for small G proteins, as a recurrent target of isoform-specific DNA hypermethylation in JMML (51% of 125 patients analyzed). RASA4 isoform 2 promoter methylation correlated with clinical parameters predicting poor prognosis (older age, elevated fetal hemoglobin), with higher risk of relapse after hematopoietic stem cell transplantation, and with PTPN11 mutation. The level of isoform 2 methylation increased in relapsed cases after transplantation. Interestingly, most JMML cases with monosomy 7 exhibited hypermethylation on the remaining RASA4 allele. The results corroborate the significance of epigenetic modifications in the phenotype of aggressive JMML.

Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome.

Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

Evolutionary dynamics of segmental duplications from human Y-chromosomal euchromatin/heterochromatin transition regions.

Human chromosomal regions enriched in segmental duplications are subject to extensive genomic reorganization. Such regions are particularly informative for illuminating the evolutionary history of a given chromosome. We have analyzed 866 kb of Y-chromosomal non-palindromic segmental duplications delineating four euchromatin/heterochromatin transition regions (Yp11.2/Yp11.1, Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2). Several computational methods were applied to decipher the segmental duplication architecture and identify the ancestral origin of the 41 different duplicons. Combining computational and comparative FISH analysis, we reconstruct the evolutionary history of these regions. Our analysis indicates a continuous process of transposition of duplicated sequences onto the evolving higher primate Y chromosome, providing unique insights into the development of species-specific Y-chromosomal and autosomal duplicons. Phylogenetic sequence comparisons show that duplicons of the human Yp11.2/Yp11.1 region were already present in the macaque-human ancestor as multiple paralogs located predominantly in subtelomeric regions. In contrast, duplicons from the Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2 regions show no evidence of duplication in rhesus macaque, but map to the pericentromeric regions in chimpanzee and human. This suggests an evolutionary shift in the direction of duplicative transposition events from subtelomeric in Old World monkeys to pericentromeric in the human/ape lineage. Extensive chromosomal relocation of autosomal-duplicated sequences from euchromatin/heterochromatin transition regions to interstitial regions as demonstrated on the pygmy chimpanzee Y chromosome support a model in which substantial reorganization and amplification of duplicated sequences may contribute to speciation.

AKAP12, a gene with tumour suppressor properties, is a target of promoter DNA methylation in childhood myeloid malignancies.

A-kinase anchor protein 12 (AKAP12) is a scaffold protein that participates in mitotic regulation and other signalling processes and probably exerts tumour suppressor function. We hypothesized that epigenetic repression of the AKAP12 gene might occur in malignant myeloid disorders. This study demonstrated that the 5' CpG island of AKAP12 was unmethylated in normal haematopoietic progenitors and granulocytes but exhibited profound methylation in Kasumi-1 and SKNO-1 leukaemic myeloblasts. Correspondingly, AKAP12 was expressed in normal progenitors but transcriptionally silent in leukaemic blasts. Re-expression of AKAP12 in Kasumi-1 and SKNO-1 cells was accomplished by treatment with MS275 alone or in combination with zebularine, indicating epigenetic mechanisms of gene repression. AKAP12 hypermethylation was found in one case of refractory anaemia with excess blasts (RAEB) and two cases of acute myeloid leukaemia (AML) in a panel of 21 blood or bone marrow samples from children with malignant myeloid disorders including refractory cytopenia, RAEB, juvenile myelomonocytic leukaemia and AML. While AKAP12 function has not been previously linked to leukaemogenesis, our results raise the possibility that epigenetic silencing of AKAP12 is involved in myeloid malignancies.