Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.

Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis.

Myelodysplastic syndromes (MDSs) are chronic and often progressive myeloid neoplasms associated with remarkable heterogeneity in the histomorphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. Recently, mutations in a gene encoding a spliceosomal protein, SF3B1, were discovered in a distinct form of MDS with ring sideroblasts. Whole exome sequencing of 15 patients with myeloid neoplasms was performed, and somatic mutations in spliceosomal genes were identified. Sanger sequencing of 310 patients was performed to assess phenotype/genotype associations. To determine the functional effect of spliceosomal mutations, we evaluated pre-mRNA splicing profiles by RNA deep sequencing. We identified additional somatic mutations in spliceosomal genes, including SF3B1, U2AF1, and SRSF2. These mutations alter pre-mRNA splicing patterns. SF3B1 mutations are prevalent in low-risk MDS with ring sideroblasts, whereas U2AF1 and SRSF2 mutations are frequent in chronic myelomonocytic leukemia and advanced forms of MDS. SF3B1 mutations are associated with a favorable prognosis, whereas U2AF1 and SRSF2 mutations are predictive for shorter survival. Mutations affecting spliceosomal genes that result in defective splicing are a new leukemogenic pathway. Spliceosomal genes are probably tumor suppressors, and their mutations may constitute diagnostic biomarkers that could potentially serve as therapeutic targets.

Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis.

Loss of heterozygosity affecting chromosome 7q is common in acute myeloid leukemia and myelodysplastic syndromes, pointing toward the essential role of this region in disease phenotype and clonal evolution. The higher resolution offered by recently developed genomic platforms may be used to establish more precise clinical correlations and identify specific target genes. We analyzed a series of patients with myeloid disorders using recent genomic technologies (1458 by single-nucleotide polymorphism arrays [SNP-A], 226 by next-generation sequencing, and 183 by expression microarrays). Using SNP-A, we identified chromosome 7q loss of heterozygosity segments in 161 of 1458 patients (11%); 26% of chronic myelomonocytic leukemia patients harbored 7q uniparental disomy, of which 41% had a homozygous EZH2 mutation. In addition, we describe an SNP-A-isolated deletion 7 hypocellular myelodysplastic syndrome subset, with a high rate of progression. Using direct and parallel sequencing, we found no recurrent mutations in typically large deletion 7q and monosomy 7 patients. In contrast, we detected a markedly decreased expression of genes included in our SNP-A defined minimally deleted regions. Although a 2-hit model is present in most patients with 7q uniparental disomy and a myeloproliferative phenotype, haplodeficient expression of defined regions of 7q may underlie pathogenesis in patients with deletions and predominant dysplastic features.

CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations and additional chromosomal aberrations constitute molecular events in chronic myelogenous leukemia.

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML.

Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A.

Chronic myelomonocytic leukemia (CMML), a myelodysplastic/myeloproliferative neoplasm, is characterized by monocytic proliferation, dysplasia, and progression to acute myeloid leukemia. CMML has been associated with somatic mutations in diverse recently identified genes. We analyzed 72 well-characterized patients with CMML (N = 52) and CMML-derived acute myeloid leukemia (N = 20) for recurrent chromosomal abnormalities with the use of routine cytogenetics and single nucleotide polymorphism arrays along with comprehensive mutational screening. Cytogenetic aberrations were present in 46% of cases, whereas single nucleotide polymorphism array increased the diagnostic yield to 60%. At least 1 mutation was found in 86% of all cases; novel UTX, DNMT3A, and EZH2 mutations were found in 8%, 10%, and 5.5% of patients, respectively. TET2 mutations were present in 49%, ASXL1 in 43%, CBL in 14%, IDH1/2 in 4%, KRAS in 7%, NRAS in 4%, and JAK2 V617F in 1% of patients. Various mutant genotype combinations were observed, indicating molecular heterogeneity in CMML. Our results suggest that molecular defects affecting distinct pathways can lead to similar clinical phenotypes.

Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. When we investigated the presence of recurrent molecular lesions in a cohort of 49 children with JMML, neurofibromatosis phenotype (and thereby NF1 mutation) was present in 2 patients (4%), whereas previously described PTPN11, NRAS, and KRAS mutations were found in 53%, 4%, and 2% of cases, respectively. Consequently, a significant proportion of JMML patients without identifiable pathogenesis prompted our search for other molecular defects. When we applied single nucleotide polymorphism arrays to JMML patients, somatic uniparental disomy 11q was detected in 4 of 49 patients; all of these cases harbored RING finger domain c-Cbl mutations. In total, c-Cbl mutations were detected in 5 (10%) of 49 patients. No mutations were identified in Cbl-b and TET2. c-Cbl and RAS pathway mutations were mutually exclusive. Comparison of clinical phenotypes showed earlier presentation and lower hemoglobin F levels in patients with c-Cbl mutations. Our results indicate that mutations in c-Cbl may represent key molecular lesions in JMML patients without RAS/PTPN11 lesions, suggesting analogous pathogenesis to those observed in chronic myelomonocytic leukemia (CMML) patients.

Heterogeneity of Cognitive Profiles in Children and Adolescents with Mild Intellectual Disability (MID).

Mild Intellectual Disability (MID) is a neurodevelopmental disorder that begins in childhood and is characterized by limitations in intellectual functioning (IQ = 55-69) and adaptive behavior that manifests in everyday living. In addition to these specific criteria, clinical practice shows that the population of children with MID has heterogeneous deficits in cognitive functioning. Thus, the aim of this study was to identify groups of homogenous cognitive profiles within a heterogeneous population of students with MID. The cognitive profiles of 16,411 participants with Mild Intellectual Disability were assessed based on their performance on the Stanford-Binet Intelligence Scales-Fifth Edition. Prior to the assessment, participants were divided into three age groups corresponding to the levels of the Polish education system: (1) 7;00-9;11, (2) 10;00-14;11, and (3) 15;00-18;11 years old. Using cluster analysis, we identified three distinct cognitive profiles (clusters) in each age group. These clusters differed from each other within and between each age group. Distinguishing cognitive profiles among children and adolescents with MID is important both in the context of diagnosis as well as the development of research-based interventions for these students.

Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms.

Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number-neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders.

Loss of expression of neutrophil proteinase-3: a factor contributing to thrombotic risk in paroxysmal nocturnal hemoglobinuria.

BACKGROUND: A deficiency of specific glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria may be responsible for most of the clinical features of this disease, but some functional consequences may be indirect. For example, the absence of certain glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria cells may influence expression of other membrane proteins. Membrane-bound proteinase 3 co-localizes with glycosylphosphatidyl inositol-linked neutrophil antigen 2a, which is absent in patients with paroxysmal nocturnal hemoglobinuria. DESIGN AND METHODS: We compared expression of proteinase 3 and neutrophil antigen 2a by flow cytometry and western blotting in normal and paroxysmal nocturnal hemoglobinuria cells and measured cytoplasmic and soluble levels of proteinase 3 by enzyme-linked immunosorbent assays in controls and patients with paroxysmal nocturnal hemoglobinuria. Finally, we studied the effects of proteinase 3 on platelet activation using an in vitro aggregometry assay and flow cytometry. RESULTS: We showed that membrane-bound proteinase 3 is deficient in patients' cells, but invariantly present in the cytoplasm regardless of disease phenotype. When we isolated lipid rafts from patients, both molecules were detected only in the rafts from normal cells, but not diseased ones. Membrane-bound proteinase 3 was associated with a decrease in plasma proteinase 3 levels, clone size and history of thrombosis. In addition, we found that treating platelets ex vivo with proteinase 3, but not other agonists, decreased the exposure of an epitope on protease activated receptor-1 needed for thrombin activation. Conversely, treatment of whole blood with serine protease inhibitor enhanced expression of this epitope on protease activated receptor-1 located C-terminal to the thrombin cleavage site on platelets. CONCLUSIONS: We demonstrated that deficiency of glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria results in decreased membrane-bound and soluble proteinase 3 levels. This phenomenon may constitute another mechanism contributing to a prothrombotic propensity in patients with paroxysmal nocturnal hemoglobinuria.

Heterogeneity of cognitive profiles in students with borderline intellectual functioning. / Heterogenicznosc profili poznawczych uczniów z inteligencja nizsza niz przecietna.

OBJECTIVES: Borderline intellectual functioning (BIF) is aclinical entity of polyetiological nature which manifests in heterogeneity of cognitive deficits. The aim of this study was to identify groups of homogenous cognitive profiles within a heterogeneous population of students with BIF. METHODS: Cognitive profiles of 114 participants with borderline intellectual functioning were assessed based on different patterns of their performance on the Wechsler Intelligence Scale for Children - Revised. RESULTS: Through a hierarchical cluster analysis we identified four distinct cognitive profiles: a) children with severe verbal skills deficits and average visual-spatial abilities; b) children with short-term memory and attention deficits; c) children with ACID profile, typical for learning disabilities; d) children with 'flat' cognitive profile where all verbal and performance skills were on borderline IQ level. CONCLUSIONS: Identifying strengthsand limitations of distinct cognitive profiles among students with borderline intellectual functioning has important implications for further assessment strategies and distinctive approach in designing educational and developmental interventions.

Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations.

Mutations in NF1, PTPN11, NRAS, KRAS and CBL have been reported to play a pathogenetic role in juvenile myelomonocytic leukaemia (JMML), a rare myelodyplastic/myeloproliferative neoplasm occurring in children. Recently, mutations in ASXL1 were identified in chronic myelomonocytic leukaemia and other myeloid malignancies. We sequenced exon 12 of ASLX1 in 49 JMML patients, and found 2 novel heterozygous (nonsense and frameshift) mutations, one occurring as a sole lesion, the other was in conjunction with a PTPN11 mutation. ASXL1 cooperates with KDM1A in transcriptional repression and thereby ASXL1 mutations may synergize with or mimic other JMML-related mutations.

Altered lipid raft composition and defective cell death signal transduction in glycosylphosphatidylinositol anchor-deficient PIG-A mutant cells.

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal disorder of haematopoietic stem cells caused by somatic PIGA mutations, resulting in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-AP). Because GPI-AP associate with lipid rafts (LR), lack of GPI-AP on PNH cells may result in alterations in LR-dependent signalling. Conversely, PNH cells are a suitable model for investigating LR biology. LR from paired, wild-type GPI(+), and mutant GPI(-) cell lines (K562 and TF1) were isolated and analysed; GPI(-) LR contained important anti-apoptotic proteins, not found in LR from GPI(+) cells. When methyl-beta-cyclodextrin (MbetaCD) was utilized to probe for functional differences between normal and GPI(-) LR, increased levels of phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-p65 nuclear factor NF-kappaB were found in control and GPI(-) cells respectively. Subsequent experiments addressing the inhibition of phosphoinositide-3-kinase (PI3K) suggest that the PI3K/AKT pathway may be responsible for the resistance of K562 GPI(-)cells to negative effects of MbetaCD. In addition, transduction of tumour necrosis factor-alpha (TNF-alpha) signals in a LR-dependent fashion increased induction of p38 MAPK in GPI(+) and increased pro-survival NF-kappaB levels in K562 GPI(-) cells. Therefore, we suggest that the altered LR-dependent signalling in PNH-like cells may induce different responses to pro-inflammatory cytokines from those observed in cells with intact GPI-AP.

Gender and Body-Fat Status as Predictors of Parental Feeding Styles and Children's Nutritional Knowledge, Eating Habits and Behaviours.

The home food environment is critically important for the development of children’s health-related practices. By managing dietary restrictions, providing nutritional knowledge and demonstrating eating behaviours, parents contribute to children’s food preferences and eating patterns. The present study examined nutritional knowledge, eating habits and appetite traits among 387 Polish five-year-old healthy and overfat boys and girls in the context of parental feeding styles and body-fat status. We observed that girls presented healthier eating habits than boys; however, overfat boys had better nutritional knowledge. Children’s body-fat percentage (%BF) was found to be linked with eating behaviours such as low satiety responsiveness and increased food responsiveness in girls as well as low emotional undereating and increased emotional overeating in boys. Our results revealed that overfat mothers, who were more prone to use the encouragement feeding style, rarely had daughters with increased %BF. Parents of overfat girls, however, were less likely to apply encouragement and instrumental feeding styles. Contrary to popular belief and previous studies, overfat women do not necessarily transmit unhealthy eating patterns to their children. Parents’ greater emphasis on managing the weight and eating habits of daughters (rather than sons) probably results from their awareness of standards of female physical attractiveness.

The potential of DNA modifications as biomarkers and therapeutic targets in oncology.

Knowledge of epigenetic alterations in cancer is rapidly increasing due to the development of genome-wide techniques for their identification. DNA methylation is the best understood epigenetic adaptation and disease-specific aberrant DNA methylation is a well-recognized hallmark of cancer. Recently, novel modifications, including 5-hydroxymethylation have been described, adding a new layer of complexity to understanding the epigenetic machinery and their role in cancer. There have been significant advances in techniques for the discovery and validation of DNA methylation- and hydroxymethylation-based biomarkers, each with its own advantages and limitations. With the advent of new profiling technologies, the ever-growing list of genes that show epigenetic alterations, particularly DNA methylation, emphasizes the role of these changes for early detection, diagnosis, prognosis, and prediction of response to therapies. While there are yet many challenges to the effective implementation of DNA-methylation/hydroxymethylation-based biomarkers and epigenetic therapeutics, the field is moving closer to the goal of defining personalized medicine.

Mutational determinants of epigenetic instablity in myeloid malignancies.

Until recently, myeloid neoplasms have been attributed to genomic and genetic instability leading to clonal outgrowth. However, it is now increasingly evident that epigenetic abnormalities also play a fundamental role in development of these malignancies. A growing body of evidence has underlined the involvement of epigenetic machinery in the malignant transformation of hematopoietic cells. Epigenetic dysfunction can lead to genetic alterations, including microsatellite instability, nucleotide changes, and chromosomal alterations. Conversely, putative epigenetic instability may be related to mutations of genes involved in epigenetic regulation. Therefore, this review focuses on epigenetic processes, including DNA methylation, post-translational histone modifications, and RNA interference via small noncoding RNAs, which play a critical role in controlling gene expression and are targets of dysregulation in many hematologic malignancies. Further, recent literature identified somatic mutations in several epigenetic regulators with a high frequency in myeloid malignancies.

Topography, clinical, and genomic correlates of 5q myeloid malignancies revisited.

PURPOSE: Interstitial deletions of chromosome 5q are common in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), pointing toward the pathogenic role of this region in disease phenotype and clonal evolution. The higher level of resolution of single-nucleotide polymorphism array (SNP-A) karyotyping may be used to find cryptic abnormalities and to precisely define the topographic features of the genomic lesions, allowing for more accurate clinical correlations. PATIENTS AND METHODS: We analyzed high-density SNP-A karyotyping at diagnosis for a cohort of 1,155 clinically well-annotated patients with malignant myeloid disorders. results: We identified chromosome 5q deletions in 142 (12%) of 1,155 patients and uniparental disomy segments (UPD) in four (0.35%) of 1,155 patients. Patients with deletions involving the centromeric and telomeric extremes of 5q have a more aggressive disease phenotype and additional chromosomal lesions. Lesions not involving the centromeric or telomeric extremes of 5q are not exclusive to 5q- syndrome but can be associated with other less aggressive forms of MDS. In addition, larger 5q deletions are associated with either del(17p) or UPD17p. In 31 of 33 patients with del(5q) AML, either a deletion involving the centromeric and/or telomeric regions or heterozygous mutations in NPM1 or MAML1 located in 5q35 were present. CONCLUSION: Our results suggest that the extent of the affected region on 5q determines clinical characteristics that can be further modified by heterozygous mutations present in the telomeric extreme.

Single nucleotide polymorphism array lesions, TET2, DNMT3A, ASXL1 and CBL mutations are present in systemic mastocytosis.

We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A) and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM). SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2 and the CBL gene family. Overall survival (OS) was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning TET2/KIT and DNMT3A. Mutations in TET2, DNMT3A, ASXL1 and CBL were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in EZH2 and IDH1/IDH2. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined TET2/DNMT3A/ASXL1 mutations independent of KIT (P = 0.04) and sole TET2 mutations (P<0.001). In conclusion, TET2, DNMT3A and ASXL1 mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations.

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T.

Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation and proliferation.

Dasatinib is an oral small molecule inhibitor of Abl and Src family tyrosine kinases (SFK), including p56(Lck) (Lck). Given the central importance of Lck in transmitting signals from the T-cell receptor (TCR) signaling complex and the potent ability of dasatinib to inhibit Lck activity, we hypothesized this agent could provide a novel route of immunomodulation via targeted inhibition of antigen-induced signaling. Herein, we show that dasatinib inhibits TCR-mediated signal transduction, cellular proliferation, cytokine production, and in vivo T-cell responses. However, dasatinib-mediated inhibition does not induce apoptosis because the effect is reversible or may be overcome by signals bypassing the TCR, such as phorbol ester. Signal transduction and proliferative responses via IL-2 remain essentially unperturbed, suggesting that dasatinib displays specificity for TCR signaling. In addition, dasatinib combined with cyclosporine A or rapamycin led to a much more potent inhibition of T-cell activation, suggesting that targeted inhibition of Lck could be a useful adjunct for enhanced immunomodulation. In combination with currently available immunomodulatory agents, SFK inhibition could potentially increase immunomodulatory efficacy while minimizing toxicity of individual agents.

Immunogenetic factors determining the evolution of T-cell large granular lymphocyte leukaemia and associated cytopenias.

T-cell large granular lymphocyte leukaemia (T-LGL) is a chronic clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGL presents with cytopenias, often accompanied by autoimmune diseases, suggesting clonal transformation arising from an initially polyclonal immune response. Various immunogenetic predisposition factors, previously described for both immune-mediated bone marrow failure and autoimmune conditions, may promote T-LGL evolution and/or development of cytopenias. The association of T-LGL was analysed with a number of immunogenetic factors in 66 patients, including human leucocyte antigen (HLA) and killer-cell immunoglobulin-like receptor (KIR) genotype, KIR/KIR-L mismatch, CTLA-4 (+49 A/G),CD16-158V/F, CD45 polymorphisms, cytokine single nucleotide polymorphisms including: TNF-alpha (-308G/A), TGF-beta1 (codons 10 C/T, 25 G/C), IL-10 (-1082 G/A), IL-6 (-174 C/G), and IFN-gamma(+874 T/A). A statistically significant increase in A/A genotype for TNF-alpha-308, IL-10-1082, andCTLA-4 +49 was observed in T-LGL patients compared with control, suggesting that the G allele serves a protective role in each case. No association was found between specific KIR/HLA profile and disease. KIR/KIR-L analysis revealed significant mismatches between KIR3DL2 and KIR2DS1 and their ligands HLA-A3/11 and HLA-C group 2 (P = 0.03 and 0.01 respectively); the biological relevance of this finding is questionable. The significance of additional genetic polymorphisms and their clinical correlation to evolution of T-LGL requires future analysis.