Incidence and prognosis of clonal hematopoiesis in patients with chronic idiopathic neutropenia.

The incidence and prognosis of clonal hematopoiesis in patients with isolated neutropenia among patients with idiopathic cytopenia of undetermined significance (ICUS), known as ICUS-N or chronic idiopathic neutropenia (CIN) patients, is poorly defined. The current study sought to investigate the frequency and clinical significance of mutations of genes implicated in myeloid malignancies using next-generation sequencing in patients with CIN (n = 185) with a long follow-up. We found that 21 (11.35%) of 185 patients carried a total of 25 somatic mutations in 6 genes with a median variant allele frequency of 12.75%. The most frequently mutated genes were DNMT3A and TET2 involving >80% of patients, followed by IDH1/2, SRSF2, and ZRSR2. The frequency of transformation to a myeloid malignancy was low in the total group of patients (5 of 185 patients [2.70%]). However, from the transformed patients, 4 belonged to the clonal group (4 of 21 [19.05%]) and 1 to the nonclonal group (1 of 164 [0.61%]), indicating that the presence of mutation(s) confers a relative risk for transformation of 31.24 (P = .0017). The variant allele frequency of the mutant clones in the transformed patients was >10% in all cases, and the genes most frequently associated with malignant transformation were SRSF2 and IDH1. No significant differences were identified between the clonal and nonclonal groups in the severity of neutropenia. Patients with clonal disease were older compared with nonclonal patients. These data contribute to the better understanding of the heterogeneous entities underlying ICUS and highlight the importance of mutation analysis for the diagnosis and prognosis of patients with unexplained neutropenias.

Significance of regional population HLA immunogenetic datasets in the efficacy of umbilical cord blood banks and marrow donor registries: a study of Cretan HLA genetic diversity.

BACKGROUND AIMS: The high genetic diversity of HLA across populations significantly confines the effectiveness of a donor or umbilical cord blood search for allogeneic hematopoietic stem cell transplantation (HSCT). This study aims to probe the HLA immunogenetic profile of the population of Crete, a Greek region with specific geographic and historical characteristics, and to investigate potential patterns in HLA distribution following comparison with the Deutsche Knochenmarkspenderdatei (DKMS) donor registry. It also aims to highlight the importance of regional public cord blood banks (PCBBs) in fulfilling HSCT needs, especially in countries with significant genetic diversity. METHODS: A cohort of 1835 samples representative of the Cretan population was typed for HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1, HLA-DPB1) loci by high-resolution second field next-generation sequencing. Data were compared with the respective HLA profiles of 12 DKMS populations (n = 20 032). Advanced statistical and bioinformatics methods were employed to assess specific intra- and inter-population genetic indexes associated with the regional and geographic distribution of HLA alleles and haplotypes. RESULTS: A considerable HLA allelic and haplotypic diversity was identified among the Cretan samples and between the latter and the pooled DKMS cohort. Even though the HLA allele and haplotype frequency distribution was similar to regions of close geographic proximity to Crete, a clinal distribution pattern from the northern to southern regions was identified. Significant differences were also observed between Crete and the Greek population of DKMS. CONCLUSIONS: This study provides an in-depth characterization of the HLA immunogenetic profile in Crete and reveals the importance of demographic history in HLA heterogeneity and donor selection. The novel HLA allele and haplotype frequency comparative data between the Cretan and other European populations signify the importance of regional PCBBs in prioritizing HLA diversity to efficiently promote the HSCT program at the national level and beyond.

Increased proportion and altered properties of intermediate monocytes in the peripheral blood of patients with lower risk Myelodysplastic Syndrome.

Immune deregulation has a critical role in the pathogenesis of lower risk myelodysplastic syndromes (MDS). The cells of the macrophage/monocyte lineage have been reported to contribute to the inflammatory process in MDS through impaired phagocytosis of the apoptotic hemopoietic cells and abnormal production of cytokines. In the present study we assessed the number of peripheral blood (PB) monocyte subsets, namely the classical CD14bright/CD16-, intermediate CD14bright/CD16+ and non-classical CD14dim/CD16+ cells, in patients with lower risk (low/intermediate-I) MDS (n = 32). We also assessed the production of tumor necrosis factor (TNF)α by patient PB monocytes in response to immune stimulus as well as their transcriptome profile. Compared to age- and sex-matched healthy individuals (n = 19), MDS patients had significantly lower number of classical and increased number of intermediate monocytes. Patient intermediate monocytes displayed increased production of TNFα following stimulation with lipopolysaccharide, compared to healthy individuals. Transcriptional profiling comparison of CD16+ monocytes from patients and controls revealed 43 differentially expressed genes mostly associated with biological pathways/processes relevant to hemopoiesis, immune signaling and cell adhesion. These data provide evidence for the first-time that distinct monocyte subsets display abnormal quantitative and functional characteristics in lower risk MDS substantiating their role in the immune deregulation associated with the disease.

Immunoglobulin and B-cell disturbances in patients with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is a granulocytic disorder associated with presence of activated, myelosuppressive T-lymphocytes. In the present study we have evaluated constituents of humoral immunity in CIN patients (n=48) compared to healthy controls (n=52). CIN patients displayed lower serum IgG levels due to a reduction in IgG1, IgG3, IgG4 but not IgG2, lower IgA and increased IgM levels compared to controls. The proportion of CD19+ cells did not differ between patients and controls; however the proportion of the naïve IgD+/CD27- B-cells was increased and the proportion of class-switched memory IgD-/CD27+ B-cells was decreased in the patients. The percentage of CD40+ B-cells did not differ between patients and controls and no aberrations in the CD40-meadiated signal transduction pathway or in CD40-gene polymorphisms were identified. These data provide further evidence that immune disturbances are associated with the pathophysiology of CIN and point out for the first time the implication of the B-cell system.

Minor populations of paroxysmal nocturnal hemoglobinuria-type cells in patients with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro-inflammatory mediators and oligoclonal/monoclonal T-lymphocytes. Because patients with immune-mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)-type cells in the peripheral blood (PB), we investigated the possible existence of PNH-type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH-type glycophorin A+ /CD59dim and glycophorin A+ /CD59- red blood cells (RBCs), FLAER- /CD24- granulocytes, and FLAER- /CD14- monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH-type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH-type cell populations. The number of patients, displaying percentages of PNH-type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T-cell receptor repertoire suggesting that T-cell-mediated immune processes underlie the emergence of PNH-type cells in CIN. Our findings suggest that patients with CIN display PNH-type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune-mediated BM failure syndromes.

Immunomodulatory actions of myeloid-derived suppressor cells in the context of innate immunity.

Myeloid-derived suppressor cells (MDSCs) are notable innate immune cells, which are further divided into two subpopulations, i.e., monocytic and granulocytic. These cells are traditionally considered to mainly suppress the T-cell responses. However, more updated data indicate that their properties are rather immunomodulatory than solely immunosuppressive. Indeed, MDSCs display extensive crosstalk with other either innate or adaptive immune cells, and, according to the situation under which they are triggered, they may enhance or attenuate the immune response. However, their positive role in host's defense mechanisms under specific conditions is rarely discussed in the literature. In this mini-review, the authors briefly summarise the mechanisms of action of MDSCs under distinct conditions, such as infections and malignancies, with a particular emphasis on their role as components of the innate immunity system.

Perspectives for the Use of Umbilical Cord Blood in Transplantation and Beyond: Initiatives for an Advanced and Sustainable Public Banking Program in Greece.

The umbilical cord blood (UCB) donated in public UCB banks is a source of hematopoietic stem cells (HSC) alternative to bone marrow for allogeneic HSC transplantation (HSCT). However, the high rejection rate of the donated units due to the strict acceptance criteria and the wide application of the haploidentical HSCT have resulted in significant limitation of the use of UCB and difficulties in the economic sustainability of the public UCB banks. There is an ongoing effort within the UCB community to optimize the use of UCB in the field of HSCT and a parallel interest in exploring the use of UCB for applications beyond HSCT i.e., in the fields of cell therapy, regenerative medicine and specialized transfusion medicine. In this report, we describe the mode of operation of the three public UCB banks in Greece as an example of an orchestrated effort to develop a viable UCB banking system by (a) prioritizing the enrichment of the national inventory by high-quality UCB units from populations with rare human leukocyte antigens (HLA), and (b) deploying novel sustainable applications of UCB beyond HSCT, through national and international collaborations. The Greek paradigm of the public UCB network may become an example for countries, particularly with high HLA heterogeneity, with public UCB banks facing sustainability difficulties and adds value to the international efforts aiming to sustainably expand the public UCB banking system.

Impaired clearance of apoptotic cells leads to HMGB1 release in the bone marrow of patients with myelodysplastic syndromes and induces TLR4-mediated cytokine production.

Excessive pro-inflammatory cytokine production in the bone marrow has been associated with the pathogenesis of myelodysplastic syndromes. We herein investigated the involvement of toll-like receptors and their endogenous ligands in the induction/maintenance of the inflammatory process in the marrow of patients with myelodysplastic syndromes. We evaluated the expression of toll-like receptors in marrow monocytes of patients (n=27) and healthy controls (n=25) by flow-cytometry and also assessed the activation of the respective signaling using a real-time polymerase chain reaction-based array. We measured the high mobility group box-1 protein, a toll-like receptor-4 ligand, in marrow plasma and long-term bone marrow culture supernatants by an enzyme-linked immunosorbent assay and we performed cross-over experiments using marrow plasma from patients and controls in the presence/absence of a toll-like receptor-4 inhibitor to evaluate the pro-inflammatory cytokine production by chemiluminescence. We assessed the apoptotic cell clearance capacity of patients' macrophages using a fluorescence microscopy-based assay. We found over-expression of toll-like receptor-4 in patients' marrow monocytes compared to that in controls; this over-expression was associated with up-modulation of 53 genes related to the respective signaling. Incubation of patients' monocytes with autologous, but not with normal, marrow plasma resulted in over-production of pro-inflammatory cytokines, an effect that was abrogated by the toll-like receptor-4 inhibitor suggesting that the pro-inflammatory cytokine production in myelodysplastic syndromes is largely mediated through toll-like receptor-4. The levels of high mobility group box-1 protein were increased in patients' marrow plasma and culture supernatants compared to the levels in controls. Patients' macrophages displayed an impaired capacity to engulf apoptotic cells and this defect was associated with excessive release of high mobility group box-1 protein by dying cells. A primary apoptotic cell clearance defect of marrow macrophages in myelodysplastic syndromes may contribute to the induction/maintenance of the inflammatory process through aberrant release of molecules inducing toll-like receptor-4 such as high mobility group box-1 protein.

Mesenchymal stem cells in immune-mediated bone marrow failure syndromes.

Immune-mediated bone marrow failure syndromes (BMFS) are characterized by ineffective marrow haemopoiesis and subsequent peripheral cytopenias. Ineffective haemopoiesis is the result of a complex marrow deregulation including genetic, epigenetic, and immune-mediated alterations in haemopoietic stem/progenitor cells, as well as abnormal haemopoietic-to-stromal cell interactions, with abnormal release of haemopoietic growth factors, chemokines, and inhibitors. Mesenchymal stem/stromal cells (MSCs) and their progeny (i.e., osteoblasts, adipocytes, and reticular cells) are considered as key cellular components of the bone marrow haemopoietic niche. MSCs may interfere with haemopoietic as well as immune regulation. Evidence suggests that bone marrow MSCs may be involved in immune-mediated BMFS underlying pathophysiology, harboring either native abnormalities and/or secondary defects, caused by exposure to activated marrow components. This review summarizes previous as well as more recent information related to the biologic/functional characteristics of bone marrow MSCs in myelodysplastic syndromes, acquired aplastic anemia, and chronic idiopathic neutropenia.

Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia.

BACKGROUND: Chronic idiopathic neutropenia is characterized by immune-mediated suppression of neutrophil production. Because patients with immune-mediated bone marrow failure syndromes display age-inappropriate telomere shortening in leukocytes, we investigated telomere lengths in peripheral blood mononuclear cells and granulocytes of patients with chronic idiopathic neutropenia. DESIGN AND METHODS: We studied 37 patients with chronic idiopathic neutropenia and 68 age- and sex-matched healthy controls. Relative telomere length and telomerase reverse transcriptase expression were assessed by a quantitative real time polymerase chain reaction. Telomerase activity was determined by a polymerase chain reaction-based immunoassay. RESULTS: The mean relative telomere values of peripheral blood mononuclear cells and granulocytes were significantly lower in patients compared to controls, and significantly lower than expected on the basis of the age-adjusted healthy control distribution. The difference in the relative telomere lengths between patients and controls in both peripheral blood mononuclear cells and granulocytes was prominent in those under the age of 50 years. Contrary to the peripheral blood mononuclear cells, in which an inverse correlation was observed between relative telomere values and age, no significant correlation was noted between granulocyte telomere values and patient age. A significant correlation was observed between individual relative telomere values and absolute neutrophil counts. There was no difference in expression of telomerase reverse transcriptase in peripheral blood mononuclear cells between patients and controls but telomerase activity was identified at a significantly higher frequency in controls than in patients. No correlation was found between telomerase activity or telomerase reverse transcriptase expression and relative telomere lengths of peripheral blood mononuclear cells. CONCLUSIONS: Patients with chronic idiopathic neutropenia display age-inappropriate telomere shortening of peripheral blood cells and low telomerase activity in peripheral blood mononuclear cells. A compensatory increased proliferation of bone marrow hematopoietic progenitor cells in association with lymphocyte replicative exhaustion probably account for these abnormalities.

Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T-lymphocytes containing T-cell receptor excision circles.

OBJECTIVES: Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T-lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T-cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T-cell lymphopenia in CIN. METHODS: We investigated parameters of T-cell homeostasis namely the proliferation/apoptotic rate of naïve and memory T cells, the T-cell senescence by telomere measurement, the recent thymic T-cell production through quantification of T-cell receptor rearrangement excision circles (TRECs), and the production of interleukin (IL)-7. RESULTS: Patients with CIN (n = 44) displayed lower proportion of naïve CD45RA(+) cells within the CD4(+) and CD8(+) cells compared with controls (n = 15). The proportion of apoptotic cells within the CD8(+) fraction was higher in patients compared with controls and was correlated with the percentage of Ki-67(+) cells, indicating an activation-induced accelerated CD8(+) cell death. The TREC content of CD4(+) and CD8(+) cells was lower in patients compared with controls and was correlated with the proportion of CD45RA(+) CD4(+) and CD8(+) cells and with the levels of serum and BM IL-7, which were significantly decreased in the patients. The mean relative telomere length of CD4(+) and CD8(+) cells was significantly lower in patients with CIN compared with age-matched controls. CONCLUSIONS: The aberrant T-cell expansions associated with the pathogenesis of CIN result in increased proliferation/apoptosis and possibly exhaustion of peripheral blood T cells which, in association with the inadequate compensatory thymic export of new TREC expressing T cells partially because of IL-7 deficiency, may contribute to lymphopenia in CIN.

Genetic associations in acquired immune-mediated bone marrow failure syndromes: insights in aplastic anemia and chronic idiopathic neutropenia.

Increasing interest on the field of autoimmune diseases has unveiled a plethora of genetic factors that predispose to these diseases. However, in immune-mediated bone marrow failure syndromes, such as acquired aplastic anemia and chronic idiopathic neutropenia, in which the pathophysiology results from a myelosuppressive bone marrow microenvironment mainly due to the presence of activated T lymphocytes, leading to the accelerated apoptotic death of the hematopoietic stem and progenitor cells, such genetic associations have been very limited. Various alleles and haplotypes of human leucocyte antigen (HLA) molecules have been implicated in the predisposition of developing the above diseases, as well as polymorphisms of inhibitory cytokines such as interferon-γ, tumor necrosis factor-α, and transforming growth factor-ß1 along with polymorphisms on molecules of the immune system including the T-bet transcription factor and signal transducers and activators of transcription. In some cases, specific polymorphisms have been implicated in the outcome of treatment on those patients.

Myeloid-Derived Suppressor Cells (MDSC) in the Umbilical Cord Blood: Biological Significance and Possible Therapeutic Applications.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells that suppress immune responses in cancer, infection, and trauma. They mainly act by inhibiting T-cells, natural-killer cells, and dendritic cells, and also by inducing T-regulatory cells, and modulating macrophages. Although they are mostly associated with adverse prognosis of the underlying disease entity, they may display positive effects in specific situations, such as in allogeneic hematopoietic stem cell transplantation (HSCT), where they suppress graft-versus-host disease (GVHD). They also contribute to the feto-maternal tolerance, and in the fetus growth process, whereas several pregnancy complications have been associated with their defects. Human umbilical cord blood (UCB) is a source rich in MDSCs and their myeloid progenitor cells. Recently, a number of studies have investigated the generation, isolation, and expansion of UCB-MDSCs for potential clinical application associated with their immunosuppressive properties, such as GVHD, and autoimmune and inflammatory diseases. Given that a significant proportion of UCB units in cord blood banks are not suitable for clinical use in HSCT, they might be used as a significant source of MDSCs for research and clinical purposes. The current review summarizes the roles of MDSCs in the UCB, as well as their promising applications.

New Perspectives on Myeloid-Derived Suppressor Cells and Their Emerging Role in Haematology.

Myeloid-derived suppressor cells (MDSCs) are immature cells of myeloid origin that have gained researchers' attention, as they constitute promising biomarkers and targets for novel therapeutic strategies (i.e., blockage of development, differentiation, depletion, and deactivation) in several conditions, including neoplastic, autoimmune, infective, and inflammatory diseases, as well as pregnancy, obesity, and graft rejection. They are characterised in humans by the typical immunophenotype of CD11b+CD33+HLA-DR-/low and immune-modulating properties leading to decreased T-cell proliferation, induction of T-regulatory cells (T-regs), hindering of natural killer (NK) cell functionality, and macrophage M2-polarisation. The research in the field is challenging, as there are still difficulties in defining cell-surface markers and gating strategies that uniquely identify the different populations of MDSCs, and the currently available functional assays are highly demanding. There is evidence that MDSCs display altered frequency and/or functionality and could be targeted in immune-mediated and malignant haematologic diseases, although there is a large variability of techniques and results between different laboratories. This review presents the current literature concerning MDSCs in a clinical point of view in an attempt to trigger future investigation by serving as a guide to the clinical haematologist in order to apply them in the context of precision medicine as well as the researcher in the field of experimental haematology.

Sputum and nasal lavage lung-specific biomarkers before and after smoking cessation.

BACKGROUND: Little is known about the effect of smoking cessation on airway inflammation. Secretory Leukocyte Protease Inhibitor (SLPI), Clara Cell protein 16 (CC16), elafin and human defensin beta-2 (HBD-2) protect human airways against inflammation and oxidative stress. In this longitudinal study we aimed to investigate changes in sputum and nasal lavage SLPI, CC16, elafin and HBD-2 levels in healthy smokers after 6 and 12 months of smoking cessation. METHODS: Induced sputum and nasal lavage was obtained from healthy current smokers (n = 76) before smoking cessation, after 6 months of smoking cessation (n = 29), after 1 year of smoking cessation (n = 22) and from 10 healthy never smokers. SLPI, CC16, elafin and HBD-2 levels were measured in sputum and nasal lavage supernatants by commercially available ELISA kits. RESULTS: Sputum SLPI and CC-16 levels were increased in healthy smokers before smoking cessation versus never-smokers (p = 0.005 and p = 0.08 respectively). SLPI and CC16 levels did not differ before and 6 months after smoking cessation (p = 0.118 and p = 0.543 respectively), neither before and 1 year after smoking cessation (p = 0.363 and p = 0.470 respectively). Nasal lavage SLPI was decreased 12 months after smoking cessation (p = 0.033). Nasal lavage elafin levels were increased in healthy smokers before smoking cessation versus never-smokers (p = 0.007), but there were no changes 6 months and 1 year after smoking cessation. CONCLUSIONS: Only nasal lavage SLPI decrease after 1 year after smoking cessation. We may speculate that there is an ongoing inflammatory process stimulating the production of counter-regulating proteins in the airways of healthy ex-smokers.

The role of CD40/CD40 ligand interactions in bone marrow granulopoiesis.

The CD40 ligand (CD40L) and CD40 are two molecules belonging to the TNF/TNF receptor superfamily, and their role in adaptive immune system has widely been explored. However, the wide range of expression of these molecules on hematopoietic as well as nonhematopoietic cells has revealed multiple functions of the CD40/CD40L interactions on different cell types and processes such as granulopoiesis. CD40 triggering on stromal cells has been documented to enhance the expression of granulopoiesis growth factors such as granulocyte-colony-stimulating factor (G-CSF) and granulocyte/monocyte-colony-stimulating factor (GM-CSF), and upon disruption of the CD40/CD40L-signaling pathway, as in the case of X-linked hyperimmunoglobulin M (IgM) syndrome (XHIGM), it can lead to neutropenia. In chronic idiopathic neutropenia (CIN) of adults, however, under the influence of an inflammatory microenvironment, CD40L plays a role in granulocytic progenitor cell depletion, providing thus a pathogenetic cause of CIN.

Two novel HLA-DRB1 alleles detected in inhabitants from the island of Crete.

Characterization of the novel HLA-DRB1*04:311 and HLA-DRB1*11:277 alleles in two Greek individuals of Cretan origin.

The Role of Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles (MSC-EVs) in Normal and Abnormal Hematopoiesis and Their Therapeutic Potential.

Mesenchymal stem cells (MSCs) represent a heterogeneous cellular population responsible for the support, maintenance, and regulation of normal hematopoietic stem cells (HSCs). In many hematological malignancies, however, MSCs are deregulated and may create an inhibitory microenvironment able to induce the disease initiation and/or progression. MSCs secrete soluble factors including extracellular vesicles (EVs), which may influence the bone marrow (BM) microenvironment via paracrine mechanisms. MSC-derived EVs (MSC-EVs) may even mimic the effects of MSCs from which they originate. Therefore, MSC-EVs contribute to the BM homeostasis but may also display multiple roles in the induction and maintenance of abnormal hematopoiesis. Compared to MSCs, MSC-EVs have been considered a more promising tool for therapeutic purposes including the prevention and treatment of Graft Versus Host Disease (GVHD) following allogenic HSC transplantation (HSCT). There are, however, still unanswered questions such as the molecular and cellular mechanisms associated with the supportive effect of MSC-EVs, the impact of the isolation, purification, large-scale production, storage conditions, MSC source, and donor characteristics on MSC-EV biological effects as well as the optimal dose and safety for clinical usage. This review summarizes the role of MSC-EVs in normal and malignant hematopoiesis and their potential contribution in treating GVHD.

The -509C/T polymorphism of transforming growth factor-beta1 is associated with increased risk for development of chronic idiopathic neutropenia.

OBJECTIVE: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro-inflammatory and pro-apoptotic mediators, such as tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and Fas-Ligand (Fas-L). In this study, we evaluated the frequency of TNF-alpha, TGF-beta1 and Fas-L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. METHODS: The TNF-alpha-308G/A, TGF-beta1 -509C/T, +869T/C, +915G/C, and Fas-L -844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well-defined area with genetically homogeneous population, using a polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS: The mutant genotype C/T or T/T of TGF-beta1 -509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild-type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18-27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF-beta1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF-alpha-308G/A, TGF-beta1 +869T/C and +915G/C and Fas-L -844T/C polymorphisms. CONCLUSIONS: The TGF-beta1 -509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF-beta1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.