Hemochromatosis-associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts.

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional iron-overload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P = 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P = 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls.

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.

Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome.

Immune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS). Immune inhibition can result from cytotoxic T cell (CTL) attack against normal hematopoiesis or reflect immune surveillance. We used clonally unique T-cell receptor (TCR) variable beta-chain (VB) CDR3 regions as markers of pathogenic CTL responses and show that while marrow failure syndromes are characterized by polyclonal expansions, overexpanded clones exist in these diseases and can serve as investigative tools. To test the applicability of clonotypic assays, we developed rational molecular methods for the detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 AA, 37 MDS, and 21 paroxysmal nocturnal hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, respectively. In total, 81 immunodominant signature clonotypes were identified. Based on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays for monitoring corresponding clones, including clonotypic Taqman polymerase chain reaction (PCR) and clonotype-specific sequencing. No correlation was found between clonality and disease severity but in patients treated with immunosuppression, truly pathogenic clones were identified based on the decline that paralleled hematologic response. We conclude that immunodominant clonotypes associated with marrow failure may be used to monitor immunosuppressive therapy.