Impact of molecular mutations on treatment response to DNMT inhibitors in myelodysplasia and related neoplasms.

We hypothesized that specific molecular mutations are important biomarkers for response to DNA methyltransferase inhibitors (DNMT inhibitors) and may have prognostic value in patients with myelodysplastic syndromes (MDS). Mutational analysis was performed in 92 patients with MDS and related disorders who received 5-azacytidine (n=55), decitabine (n=26) or both (n=11). Mutational status was correlated with overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) by univariate and multivariate analysis. Risk stratification models were created. TET2, DNMT3A, IDH1/IDH2, ASXL1, CBL, RAS and SF3B1 mutations were found in 18, 9, 8, 26, 3, 2 and 13% of patients, respectively. In multivariate analysis, TET2(MUT) and/or DNMT3A(MUT) (P=0.03), platelets > or = 100 × 10(9)/l (P=0.007) and WBC<3.0 × 10(9)/l (P=0.03) were independent predictors of better response. TET2(MUT) and/or DNMT3A(MUT) (P=0.04) status was also independently prognostic for improved PFS, as were good or intermediate cytogenetic risk (P<0.0001), age<60 (P=0.0001), treatment with both 5-azacytidine and decitabine (P=0.02) and hemoglobin > or = 10 g/dl (P=0.01). Better OS was associated with ASXL1(WT) (P=0.008) and SF3B1(MUT) (P=0.01), and, similar to PFS, cytogenetic risk (P=0.0002), age (P=0.02) and hemoglobin (P=0.04). These data support the role of molecular mutations as predictive biomarkers for response and survival in MDS patients treated with DNMT inhibitors.

Double-negative regulatory T cells induce allotolerance when expanded after allogeneic haematopoietic stem cell transplantation.

Double-negative (DN) regulatory T cells (Tregs) are specialized T lymphocytes involved in the down-modulation of immune responses, resulting in allotolerance after allogeneic haematopoietic stem cell transplantation (HSCT). Most of the properties of DN Tregs were identified in murine models, including the unique ability to suppress alloreactive syngeneic effector T cells in an antigen-specific manner via Fas/Fas-ligand interactions. We investigated the behaviour of DN Tregs following human allogeneic HSCT with regard to occurrence of graft-versus-host disease (GvHD) and restoration of T-cell receptor repertoire in a cohort of 40 patients. The frequency of DN Tregs and CD4/CD8 TCR repertoire was measured serially and at the time of diagnosis of GvHD by flow cytometry. Analysis demonstrated a positive correlation between degree of alloreactivity, as measured by grade of GvHD, and the number of variable beta chain (Vbeta) family expansions in both T-cell populations. We also found that a deficiency of DN Tregs was associated with an increased number of Vbeta family expansions, and most importantly, with the occurrence of GvHD. All individuals who demonstrated more than 1% DN Tregs did not develop GvHD, providing evidence that DN Tregs participate in peripheral tolerance to prevent GvHD when expanded after allogeneic HSCT.

Alpha satellite DNA variant-specific oligoprobes differing by a single base can distinguish chromosome 15 homologs.

The ability to distinguish homologous chromosomes is a powerful cytogenetic tool. However, traditional techniques can only distinguish extreme physical variants and are highly dependent on sample preparation. We have previously reported oligonucleotide probes, specific for human chromosome 17 alpha satellite DNA sequence variants, that distinguish cytogenetically normal homologous chromosomes by FISH. Here we report the development of similar oligoprobes, differing at a single nucleotide position, that not only distinguish homologous chromosomes 15 but can be used to follow the transmission of a chromosome from parents to their offspring. We also identified a novel array-size polymorphism in another family. The alphoid array of one chromosome is quite small and below the detection threshold for our oligoprobes, although it is detectable by conventional FISH probes. This size polymorphism provides an additional FISH-based method for distinguishing homologs. Most importantly, this work illustrates the potential applicability of the technique to the entire human chromosome complement.

Molecular cloning of the RNA polymerase I transcription factor hUBF/NOR-90 (UBTF) gene and localization to 17q21.3 by fluorescence in situ hybridization and radiation hybrid mapping.

The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to human upstream binding factor (hUBF), which has two molecular mass forms of 89 and 93 kDa, respectively. hUBF/NOR-90 is a member of the HMG-box DNA-binding protein family and is known to bind to enhancer regions upstream of the ribosomal RNA genes, which are clustered at NORs. The smaller version of UBF lacks an internal 111-bp region corresponding to 37 amino acids in the second HMG-box of the larger form. We isolated human genomic clones from a phage library and localized one of them by fluorescence in situ hybridization to chromosome 17q21.3. DNA sequence analysis showed that the 111-bp region represented a single exon, consistent with the previous notion that the two isoforms were products of alternative pre-mRNA splicing of a single gene in human. Radiation hybrid mapping placed this STS with very high probability (LOD > 19) to chromosome 17, approximately 3.77 cR distal to MIT framework marker UTR-9641. The order of the markers (a partial list) from this region was UTR-9641, SGC30031, WI-17308, D17S930, NOR53/33, WI-16100, D17S920, WI-16913, and WI-6808.

Alphoid variant-specific FISH probes can distinguish autosomal meiosis I from meiosis II non-disjunction in human sperm.

Over the past few years, several groups have used fluorescence in situ hybridization (FISH) to study aneuploidy in human sperm. Several important observations have derived from these studies, including the demonstration of chromosome-specific variation in non-disjunction frequencies, and the possible association of aneuploidy with environmental agents and with increasing paternal age. However, an important technical limitation of these studies has been the inability to distinguish between autosomal non-disjunction occurring at meiosis I and meiosis II. In the present report, we describe a simple FISH-based approach designed to overcome this limitation. Using oligonucleotide probes capable of distinguishing subtle differences in the alpha satellite sequences of chromosome 17, we demonstrate that (in appropriate heterozygotes) it is possible to simultaneously identify disomic sperm and to determine the meiotic stage of origin of the additional chromosome. This novel approach has important implications for future FISH sperm studies, since the ability to distinguish between meiosis I and meiosis II non-disjunction will make it possible to determine whether putative etiological agents affect chromosome segregation at both, or only one, of the two meiotic stages.

Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISH.

Chromosomal heteromorphisms have been used extensively to mark individual chromosomes. However, classical banding techniques used to identify these structural variants are imprecise and difficult to quantify. Different chromosomes 17 from the human population are characterized by distinct haplotypes of alpha satellite DNA. We have used these sequence variants to construct oligonuoleotide probes for fluorescence in situ hybridization (FISH). These oligomers are the first reported FISH probes that can discriminate between cytogenetically indistinguishable chromosome homologues. They have been used to follow the transmission of a single chromosome 17 through a pedigree, similar to a typical polymorphic marker. Furthermore, extended chromatin fiber techniques reveal the presence of discrete domains of different sequence variants within individual centromeres. Extension of this strategy to create a battery of other variant-specific oligoprobes should provide a powerful diagnostic tool for parent of origin effects in the study of aneuploidy, imprinting and cancer cytogenetics.

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.

Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function.

The D-type cyclin-dependent kinases CDK4 and CDK6 are complexed with many small cellular proteins (p14, p15, p16, p18, and p20). We have isolated cDNA sequences corresponding to the MTS2 genomic fragment that encodes the CDK4- and CDK6-associated p14 protein. By use of a yeast interaction screen to search for CDK6-interacting proteins, we have also identified an 18-kD human protein, p18, that is a homolog of the cyclin D-CDK4 inhibitors p16 (INK4A/MTS1) and p14 (MTS2/INK4B). Both in vivo and in vitro, p18 interacts strongly with CDK6, weakly with CDK4, and exhibits no detectable interaction with the other known CDKs. Recombinant p18 inhibits the kinase activity of cyclin D-CDK6. Distinct from the p21/p27 family of CDK inhibitors that form ternary complexes with cyclin-CDKs, only binary complexes of p14, p16, and p18 were found in association with CDK4 and/or CDK6. Ectopic expression of p18 or p16 suppresses cell growth with a correlated dependence on endogenous wild-type pRb.