Myelodysplastic syndromes (MDS) represent a heterogeneous group of neoplastic hematopoietic disorders. Several recurrent chromosomal aberrations have been associated with MDS, but the genes affected have remained largely unknown. To identify relevant genetic lesions involved in the pathogenesis of MDS, we conducted SNP array-based genomic profiling and genomic sequencing in 102 individuals with MDS and identified acquired deletions and missense and nonsense mutations in the TET2 gene in 26% of these individuals. Using allele-specific assays, we detected TET2 mutations in most of the bone marrow cells (median 96%). In addition, the mutations were encountered in various lineages of differentiation including CD34(+) progenitor cells, suggesting that TET2 mutations occur early during disease evolution. In healthy tissues, TET2 expression was shown to be elevated in hematopoietic cells with highest expression in granulocytes, in line with a function in myelopoiesis. We conclude that TET2 is the most frequently mutated gene in MDS known so far.

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

The pattern of X-chromosome inactivation (XCIP), or Lyonization, can be used to distinguish monoclonal from polyclonal cell populations in females. However, a skewed XCIP exists in hematopoietic cells in approximately 40% of healthy elderly females, interfering with interpretation of clonality assays. In hematopoiesis, an active stem cell pool is assumed to be present within a larger population of inactive stem cells, with a continuous exchange of cells between the two compartments. The assumption that the active stem cell pool size decreases with age may explain the phenomenon of acquired skewing occurring by chance and predicts the XCIP of this population to fluctuate. This fluctuation should be reflected in the XCIP of peripheral granulocytes. We examined the XCIP for fluctuations in time in peripheral granulocytes, monocytes and T cells of young, middle-aged and elderly healthy females. We used an optimized HUMARA PCR assay that eliminates unbalanced DNA amplification. We found no fluctuations in XCIP in any age group in up to 18 months follow-up. We conclude that acquired skewing arises gradually in life without fluctuations in XCIP and that analysis at multiple time points cannot distinguish monoclonal hematopoiesis from normal, skewed hematopoiesis.

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