We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional ironoverload. 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 5 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 5 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. Am. J. Hematol. 82:1076Hematol. 82: -1079Hematol. 82: , 2007
Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) have a common pathogenesis related to an immune attack on hematopoietic progenitor and stem cells. While the inciting events are not known, it is possible that complex immunogenetic predisposition factors exist which, in the context of exogenous influences, determine risk for these diseases. They include HLA background, KIR genotype, cytokine and immunomodulatory gene polymorphisms as well as gene variants involved in peptide processing and presentation. We have empirically selected a number of important polymorphisms that were described in the context of various immunologic diseases and studied their frequency in a large cohort (N=57) of PNH patients. As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of Nk-cells and cytotoxic T lymphocytes, we examined the combined impact of KIR/KIR-L genotypes on the risk of PNH and PNH/AA syndrome. PNH showed a decreased frequency of inhibitory KIR-2DL1 and KIR-2DL3 genes (79% vs. 95%, p=.0054; 67% vs. 89%, p=.0032). Analysis of the KIR genotype in correlation with the corresponding KIR-L profile, deduced from HLA typing, revealed an increased frequency of unopposed 2DS2 (2DS2/C1 type, 37% vs.10%, p=.012) and 2DL2 (2DL2/C1 mismatch 37% vs 11%, p=.031) but these mismatches have a potentially opposing functional effect. Using sequence-specific PCR amplification and/or direct sequencing, we have genotyped DNA samples derived from our cohort who presented with different subtypes of PNH and studied single nucleotide polymorphisms (SNPs) in cytokine genes such as TNF-a (−308 G/A), TGF-b 1 (C/T codon 10, C/G codon 25), IL-10 (−1082 G/A), and IFN-g (+874 A/T) and immunomodulatory receptor genes like CTLA-4 exon 6 (+49 G/A), FcγRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). These SNPs are responsible for intrinsic differences in cytokine production and receptor function and can thereby influence immune physiologic and pathologic responses. PNH patients showed a significantly higher frequency of A/A polymorphisms at intron −1082 of the IL-10 gene consistent with a “low secretor phenotype” (36% PNH (N=50) vs 13.8% controls (N=363), p=0.0001). In contrast to a few smaller studies, no association with any other SNP tested was found. To establish whether this finding translates into functional consequences, we examined basal and PMA-induced IL-10 secretion in PNH patients andcontrols. Upon induction, IL-10 production increased in controls (N=5, 14.11± 9.8), while in general, stimulation resulted in a much weaker IL-10 response in PNH patients (N=6, 4.51± 2.16). However, when compared genotypic IL-10 “low secretors” (−1082 A/A) among PNH patients showed significantly lower induction levels of IL-10 (N=2, 3.1± 1.58) as compared to PNH patients with “normal/high secretor” genotypes (−1082 A/G,G/G) ((N=4, 5.22 +/−3.1). The low secretor IL-10 genotype may correspond to the exaggerated TH1 response in PNH and in general supports the notion that complex inherited traits may exist that genetically determine propensity to PNH evolution.
Complex interaction between a multitude of genetic variants may be responsible for differential susceptibility to specific diseases, and be responsible for phenotypic variability and heterogeneity of clinical presentations. Such a variability in clinical features confounded for many years investigations into the pathogenesis of myelodysplastic syndromes (MDS). We made a curious observation of increased ferritin levels in some newly diagnosed patients with MDS RARS (refractory anemia with ringed sideroblasts) in whom transfusional iron-overload was unlikely due to very low transfusion burden. Hence, we hypothesized that RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this particular subset of MDS. We studied a cohort of 109 MDS patients; 42 with RARS, and 67 with other forms of MDS (18 RA, 12 RAEB, 7 RAEB-T, 1 CMML, and 29 MDS/MPD overlap). All patients were genotyped using restriction fragment length polymorphism (RFLP) method, designed to detect presence of C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygozity for the C282Y mutation in 21% of RARS patients (vs 9% in control population, n=2016, p= 0.017) while H63D genotype was not increased. The possible pathogenic role of this finding in RARS was supported by the normal distribution of mutant HFE alleles in patients with other forms of MDS (5% vs. 9%, p =0.35). Interestingly, 3/7 patients with RA not fulfilling the RARS criteria, but having increased numbers of ringed sideroblasts (<15%) also showed heterozygozity for either C282Y or H63D allele. To correlate the presence of C282Y allele with clinical features of RARS patients, we have performed a subset analysis. Within this group we have included patients with a rather nebulous and rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt); 7 of these patients (n=10) were found to have either C282Y or H63D allele resulting in a frequency of 30% and 40% of C282Y or H63D allele, respectively. The combined prevalence of either of these alleles in the control population is 33% (vs. 70% in RARSt, p=.01). Previously, we have demonstrated that RARSt patients are characterized by a high prevalence of the V617F JAK2 mutation (Szpurka et al, Blood 2006) suggestive of the pathophysiologic derivation of this syndrome from MPD rather than MDS. Consequently, we have tested the frequency of HFE gene variants associated with hemochromatosis in patients with MPD and Jak2 mutations. Of note is that patients with RARS harbored more C282Y alleles than those with other forms of MDS or MPD with Jak2 mutation (except for those with RARSt; (21% vs 5% and 3%, p =0.036 and .012, respectively). We conclude that hemochromatosis associated mutations may contribute to the pathogenesis of RARS. In patients with MPD and Jak2 mutation, concomitant presence of hemachromatosis-predisposing HFE variants may result in the unusual presentation associated with ringed sideroblasts.
Introduction: Chronic myelomonocytic leukemia (CMML) is a heterogeneous group of bone marrow disorders currently grouped into the MDS/MPD overlap by WHO classification. Its clinical and laboratory features includes the presence of up to 20% blast in the bone marrow and the peripheral blood, a PB monocyte count >1000/mL, splenomegaly, variable reticulin fibrosis in the bone marrow and cytopenias. The exact pathogenesis remains in question and there are no effective therapies. Recent studies in certain myeloid disorders suggest that the nuclear transcription factor NFkB regulates cell survival, proliferation, and differentiation. It has been found to be highly expressed in AML and may serve as an important therapeutic target. Little is known about NFkB in other hematologic disorders, including CMML. Examination of NFkB activation in situ has been technically difficult due to lack of quality antibody reagents suitable for fixed tissues. Recently, phosphospecific antibodies have become available, which reflect the functional status of proteins. IkB regulates NFkB subunits by sequestering them in the cytoplasm. Phosphorylation of IkB by IKK results in release of NFkB and translocation to the nucleus. Thus, phospho-IkB (pIkB) is an indicator of NFkB activation. We studied the pattern of pIkB expression in CMML as a surrogate of NFkB activation. Methods: We identified a cohort of 24 CMML (CMML1=17; CMML2=7) patients and 9 healthy controls. Cases were characterized clinically and pathologically. Immunohistochemistry (IHC) for pIkB was performed in trephine biopsies using a phosphor-specific antibody (Cell Signaling). The staining pattern was compared to normal bone marrow. We utilized JMP 5.1.2 statistical software to compare a variety of clinical and laboratory parameters. Results: The mean age of patients at diagnosis was 61 (range:38–72). Median WBC, Hgb, PLT, absolute monocytes were 18.6K/ul, 10.2g/dL, 93K/ul, 4K/ul respectively. As expected, the overall survival (OS) was short (mean OS = 11.1 months). Compared to normal bone marrow, pIkB was found to be abnormally activated in maturing granulocytic cells and was found to be present in cytoplasmic as well as nuclear locations. The mean % neutrophils that expressed pIkB was 36.6 compared to 14.9 for normal bone marrow (P<0.00001). No clear association between OS or PFS was detected in this relatively small series based on neutrophil pIkB expression. However, neutrophil pIkB expression was correlated with higher WBC (R = 0.9, P=0.03) and absolute monocyte count (R =0.19, P=.04). Conclusions: An abnormal in situ pIkB expression pattern is present in CMML compared to normal bone marrow, suggesting abnormal activation of NFkB. Interestingly, maturing myeloid cell expression of pIKB was associated with higher WBC and absolute monocyte count. Further studies are warranted in examining the role of NFkB activation in CMML and potential therapeutic intervention in this pathway, such as with proteasome inhibitors.
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