Granulocyte function in patients with L-ferritin iron-responsive element (IRE) 39C-->T-positive hereditary hyperferritinaemia-cataract syndrome.

BACKGROUND: Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant trait associated with mutations in the iron responsive element (IRE) of the ferritin light-chain (L-ferritin) gene. Patients typically show elevated serum ferritin concentrations without iron overload and a bilateral cataract. Hyperferritinaemia can be associated with granulocyte dysfunction in patients with thalassemia beta and in haemodialysis patients. The effect of increased L-ferritin levels on granulocyte function in patients with HHCS is unknown. MATERIAL AND METHODS: We examined glucose uptake, oxidative burst, chemotaxis, phagocytosis, apoptosis and intracellular calcium concentrations in polymorphonuclear leucocytes (PMNLs) of five affected members of a family with HHCS and in five healthy individuals matched for age and gender. RESULTS: Mutation testing revealed a 39C-->T transition in IRE in all five patients with HHCS. Serum ferritin levels of patients ranged between 907 and 2030 microg L(-1), respectively. In comparison with healthy individuals, PMNLs of patients with HHCS showed a significant increase in PMA-mediated stimulation of the oxidative burst, as well as a significantly higher stimulation of glucose uptake but no difference with respect to chemotaxis, phagocytosis, apoptosis and intracellular calcium concentrations. CONCLUSION: In summary, our study suggests that hyperferritinaemia in patients with IRE 39C-->T-positive HHCS is associated with activation of PMNLs but not with disturbance of fundamental PMNL function.

Numbers of colony-forming progenitors in patients with systemic mastocytosis: potential diagnostic implications and comparison with myeloproliferative disorders.

BACKGROUND: An increase in colony-forming progenitor cells (CFU) is typically seen in myeloproliferative disorders (MPD). Systemic mastocytosis (SM) is a haemopoietic neoplasm involving myeloid progenitors similar to MPD. In the present study, we measured the levels of peripheral blood (pb) and bone marrow (bm) CFU in patients with different categories of SM, and compared them with those obtained in MPD patients and healthy controls. MATERIALS AND METHODS: Numbers of CFU (CFU-GM, BFU-E, CFU-GEMM) were measured in a colony assay in 25 patients with SM [indolent SM (ISM), n = 15; smouldering SM (SSM), n = 3; SM with an associated haematologic clonal non-mast cell lineage disease (SM-AHNMD), n = 5; aggressive SM (ASM), n = 1; mast cell leukaemia (MCL), n = 1] and 37 with MPD [chronic myeloid leukaemia (CML), n = 10; polycythemia vera (PV), n = 8; essential thrombocytosis (ET), n = 9; idiopathic myelofibrosis (IMF), n = 10]. RESULTS: In the patients with MPD, elevated numbers of pb CFU were detected in all groups when compared with healthy controls (P < 0.05). In most of the patients with ISM, circulating CFU levels (CFU-GM, BFU-E, and CFU-GEMM) were within the normal range. In SSM, pb CFU-GM levels were normal in two patients, and elevated in a third patient. In the "SM-AHNMD-group", CFU levels were found to reflect the nature of the AHNMD: in SM with concomitant acute myeloid leukaemia (SM-AML, n = 2), the levels of CFU were low or undetectable, whereas in SM with chronic myelomonocytic leukaemia (SM-CMML, n = 2), elevated numbers of pb CFU-GM were found. CONCLUSION: The numbers of CFU are normal in patients with ISM, but elevated in some patients with SSM and SM-CMML. An elevated CFU level in SM should raise the suspicion of an associated MPD (CMML) or smouldering SM, a novel SM-subtype that shares several features with MPD and sometimes progresses to an overt SM-MPD.

Increased prevalence of combined MTR and MTHFR genotypes among individuals with severely elevated total homocysteine plasma levels.

The prevalence of the methionine synthase (MTR) 2756A-->G polymorphism among individuals with severely elevated total homocysteine (tHcy) plasma levels is unknown. Therefore, 1,716 subjects, including 415 hemodialysis patients, 179 peritoneal dialysis patients, 733 kidney graft recipients, and 389 healthy subjects, were investigated. The distribution of MTR 2756A-->G, as well as 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T/1298A-->C, genotypes among study participants with extremely high tHcy plasma levels (>90th percentile) was compared with the genotype distribution of subjects with very low tHcy plasma levels (<10th percentile). The prevalence of MTR 2756AG and GG genotypes alone did not differ between individuals with extremely high or extremely low tHcy levels (P = 0.7402; odds ratio [OR], 1.076; 95% confidence interval [CI], 0.697 to 1.662). Conversely, combined MTR and MTHFR genotypes (MTR 2756AG and 2756GG and MTHFR 677TT/1298AA and 677CT/1298AC) were found more often in the highest (n = 34) compared with the lowest plasma tHcy decile (n = 19; P = 0.0252; OR, 1.983; 95% CI, 1.079 to 3.643). The number of patients with the wild-type MTR and MTHFR genotype was three times greater in the lowest compared with the highest decile (17 versus 6 patients, respectively; P = 0.0155; OR, 0.330; 95% CI, 0.126 to 0.861). In summary, our study shows that the 2756A-->G transition of MTR in combination with MTHFR 677TT/1298AA and 677CT/1298AC can be associated with extremely high tHcy plasma levels.

Mutation analysis of C-KIT in patients with myelodysplastic syndromes without mastocytosis and cases of systemic mastocytosis.

The proto-oncogene C-KIT encodes a tyrosine kinase receptor that is expressed on mast cells and haematopoietic stem cells and can show somatic mutations in patients with mastocytosis. Only scattered information is available about mutations in C-KIT in patients with other myeloid neoplasms. Moreover, the prevalence of mutations in C-KIT in bone marrow specimens of individuals with systemic mastocytosis is largely unknown. Using sequence analysis, we have screened cDNAs of the C-KIT domain encompassing codon 510-626 and codon 763-858 in bone marrow (BM) mononuclear cells (MNCs) of patients with myelodysplastic syndromes (n = 28) and patients with systemic mastocytosis (n = 12) for the presence of mutations. Furthermore, restriction fragment length polymorphism analysis was applied for identification of the C-KIT 2468A-->T and the C-KIT 1700T-->G mutation, as well as the C-KIT 1642A-->C polymorphism. All 11 patients with systemic indolent mastocytosis tested positive for C-KIT 2468A-->T. In contrast, no mutation was identified in the case of aggressive mastocytosis. Among patients with myelodysplastic syndromes, no patient showed a somatic mutation in C-KIT. The allele frequency for C-KIT 1642A-->C among the entire patient population was 0.038 and was 0.125 among age- and sex-matched healthy controls. Our data demonstrate that myelodysplastic syndromes without histological or cytological evidence of mastocytosis do not exhibit somatic mutations in exons 10, 11, 12, 16, 17 and 18 of C-KIT. In contrast, BM MNCs of patients with systemic indolent mastocytosis were all positive for C-KIT 2468A-->T and negative for additional mutations in these exons. The C-KIT 1642A-->C polymorphism is not associated with myelodysplastic syndrome or systemic mastocytosis.

A case of 'smouldering' mastocytosis with high mast cell burden, monoclonal myeloid cells, and C-KIT mutation Asp-816-Val.

Mastocytosis is a term used for a group of disorders characterized by abnormal growth and accumulation of tissue mast cells (MC) in one or more organ systems. In patients with systemic mastocytosis (SM) the clinical course may be indolent or aggressive or even complicated by leukemic progression or an associated clonal hematologic non mast cell lineage disease (AHNMD). However, at first presentation (diagnosis) it may be difficult to define the category of disease and the prognosis. We report on a 48-year-old female patient with SM with urticaria pigmentosa-like skin lesions and mediator-related symptoms. She was found to have splenomegaly, a high infiltration grade (MC) in bone marrow biopsies (>30%), mild anemia, and a high serum tryptase level (>500 ng/ml). In addition, she exhibited discrete histologic signs of myeloproliferation in the 'non-affected' marrow and monoclonal blood cells established by C-KIT 2468A-->T mutation (Asp-816-Val) -analysis and HUMARA assay. Despite these findings, however, the clinical course was stable over years and no AHNMD or organ impairment developed. Because of the 'intermediate' clinical signs and absence of progression to aggressive disease, we proposed the term 'smouldering mastocytosis'.

Erythropoietin-inducible immediate-early genes in human vascular endothelial cells.

BACKGROUND: Patients receiving recombinant human erythropoietin (rHuEPO) may experience side effects arising from the vascular system. The underlying mechanisms, however, are largely unknown. METHODS: To elucidate downstream events following erythropoietin receptor triggering, a differential display analysis of human vascular endothelial cell mRNA was performed. RESULTS: We identified eight genes that were upregulated by rHuEPO as confirmed in two further independent cell culture experiments using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) protocol. The genes coded for proteins that may be assigned to four different groups: 1) proteins implicated in the regulation of vascular functions (thrombospondin-1, 20 kDa myosin regulatory light chain; relative increase of rHuEPO induced mRNA levels: 155.2%, P = 0.043; 137.6%, P = 0.046, respectively); 2) gene products involved in gene transcription and/or translation (c-myc purine-binding transcription factor PuF, tryptophanyl-tRNA synthetase, S19 ribosomal protein; increase of mRNA levels: 126.4%, P = 0.032; 150.9%, P = 0.012; 134.9%, P = 0.038); 3) subunits of mitochondrial enzymes related to energy transfer (NADH dehydrogenase subunit 6, cytochrome C oxidase subunit 1; increase of mRNA concentrations: 141.7%, P = 0.007; 140.3%, P = 0.01); and 4) regulators of signal transduction (protein tyrosine phosphatase G1, increase of transcript level: 160.3%, P = 0.016). CONCLUSIONS: We report on novel molecular downstream events following rHuEPO receptor triggering of human vascular endothelial cells. We identified EPO-responsive immediate-early genes, coding for proteins involved in vascular functions, gene transcription, and/or translation, energy transfer, and signal transduction. Thus, our data provide new insights into the molecular changes induced by EPO in human vascular endothelial cells.

Molecular analysis of the carboxy terminus of the beta and gamma subunits of the epithelial sodium channel in patients with end-stage renal disease.

BACKGROUND: Mutations in the carboxy termini of the beta subunit (hbetaENaC) and the gamma subunit (hgammaENaC) of the human epithelial sodium channel have been identified in patients with Liddle syndrome. Moreover polymorphisms have been described in these genes, the clinical relevance of which for progression to end-stage renal disease (ESRD) is unknown. We, therefore, have screened ESRD patients for putative variants of these genes. METHODS: We investigated 256 chronic hemodialysis patients, including 123 patients with a history of hypertension as a cause of ESRD. Screening for mutations in the carboxy termini of hbetaENaC and hgammaENaC was accomplished by polymerase chain reaction amplification followed by single-strand conformation polymorphism analysis. RESULTS: In 231 patients single-strand conformation polymorphism analysis of the polymerase chain reaction fragments of the hbetaENaC and hgammaENaC genes showed a similar migration pattern as compared with negative control subjects. In 25 patients a band shift was observed. However, sequence analysis in all these patients revealed wild-type sequence. CONCLUSIONS: The present study demonstrates the absence of genetic variants in the carboxy terminus of the hbetaENaC and hgammaENaC genes in Austrian patients with ESRD maintained on chronic hemodialysis treatment. Thus, mutations in these genes are unlikely to be associated with ESRD.

Systemic mastocytosis associated with acute myeloid leukaemia: report of two cases and detection of the c-kit mutation Asp-816 to Val.

A subset of patients with systemic mastocytosis (SM) develop acute myeloid leukaemia (AML). However, little is known about the biology of such leukaemias and their relationship to the mast cell (MC) lineage. We report on two female patients who suffered from SM and AML. According to FAB criteria, the leukaemias were classified as AML-M4 (patient 1) and AML-MO (patient 2). The coexistence of the two distinct neoplasms (AML and SM) was demonstrable by immunostaining of serial bone marrow (BM) sections with monoclonal antibodies (mAb). In particular, the MC infiltrates were found to react with mAb against MC-tryptase and MC growth factor receptor c-kit (CD117), but not with mAb to CD15 or CD34. In contrast, the AML blasts were immunoreactive for CD15 (patient 1) or CD34 (patient 2), but did not express tryptase. The c-kit point mutation Asp-->Val at codon 816, considered to play a role in the transformation of MC progenitors, was detected in patient 1 in a BM cell fraction containing 4% MC. However, no c-kit mutation was found in pure AML blasts (<1% MC). These findings argue against an evolution of the AML clone from neoplastic MC or MC-committed progenitors.