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Homocysteine is implicated to be an atherogenic amino acid and has been associated with increased risk of adverse cardiovascular outcomes. The prognostic significance of plasma total homocysteine (tHcy) levels for mortality and allograft loss in kidney transplant recipients has not been established. A total of 733 kidney transplant recipients who were seen for a routine visit at this transplant clinic in 1996 to 1998 were studied prospectively. During that visit, clinical information was collected and blood was drawn for laboratory evaluation. Information on the previous transplant procedure and the organ donor was obtained from the Eurotransplant Foundation database. Patients were followed prospectively using the Austrian Dialysis and Transplant Registry. With the use of proportional-hazards regression, the independent relations of fasting plasma tHcy levels to the risk of death from any cause and kidney allograft loss were examined. During a median follow-up of 6.1 yr, 154 participants died and 260 kidney allografts were lost. After adjustment for several important risk factors, elevated tHcy levels (/=12 micromol/L) were associated with 2.44 times the mortality risk of patients with normal tHcy levels (hazards ratio 2.44; 95% confidence interval 1.45 to 4.12; P0.001). Similarly, elevated tHcy levels were associated with 1.63 times increased risk of kidney allograft loss (hazards ratio 1.63; 95% confidence interval 1.09 to 2.44; P = 0.02). In this single-center sample, baseline fasting plasma tHcy levels were independently associated with the risk of death and kidney allograft loss. The clinical utility of homocysteine-lowering therapy, such as multivitamin therapy, to reduce the rates of these end points needs to be studied.

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 39CT transition in IRE in all five patients with HHCS. Serum ferritin levels of patients ranged between 907 and 2030 µg 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 39CT-positive HHCS is associated with activation of PMNLs but not with disturbance of fundamental PMNL function.

Background: In contrast to aggressive mastocytosis, patients with indolent systemic mastocytosis (ISM) usually present with urticaria pigmentosa-like skin lesions. In those who lack skin lesions, mastocytosis is often overlooked or confused with endocrinologic, allergic, or other internal disorders. Case Report and Results: We report on a 33-year-old male patient in whom severe hypotensive episodes occurred after contact with ants or yellow jackets. Since no specific IgE was detected, the serum tryptase concentration was measured and found to be clearly elevated (70 ng/ml). Consecutive staging and examination of the bone marrow revealed ISM. The patient was advised to circumvent insect contact, to take antihistamines on demand, and to carry an epinephrine self-injector for emergency events. In a retrospective analysis of 40 patients seen between 1988 and 2003, only 2 had a life-threatening mediator-related episode before ISM was diagnosed. Conclusions: Our report confirms the diagnostic value of tryptase in patients with suspected mastocytosis. In addition, the report suggests that the lack of typical skin lesions does not exclude an indolent form of mastocytosis even if the serum tryptase is clearly elevated. Finally, our case further shows that mastocytosis can be an important differential diagnosis to be considered in patients with unexplained anaphylactoid or other mediator-related symptoms.

Anderson-Fabry disease is a rare lysosomal storage disorder. It results from a deficiency of the lysosomal alpha-galactosidase A and leads to progressive accumulation of globotriaosylceramide in the endothelium and tissue cells of various organs. Some of the typical clinical findings such as tiredness, dry skin, myalgia and arthralgia as well as vague gastrointestinal complaints are also symptoms of hypothyroidism. Therefore, we studied the thyroid function in patients with Anderson-Fabry disease. Thyroid function was studied in 11 patients (6 female, 5 male) with Anderson-Fabry disease by measuring thyroid-stimulating hormone (TSH) and free thyroxine serum levels. Nine patients had chronic kidney disease with stage 1 and two with stage 5. Subclinical hypothyroidism (normal serum free thyroxine concentrations along with elevated serum TSH levels) was found in 4 of 11 patients (36.4%). Subclinical hypothyroidism was observed in both male and female patients as well as in patients with stage 1 and stage 5 kidney disease. Subclinical hypothyroidism is a common finding in patients with Anderson-Fabry disease, showing an excess prevalence as compared to the normal population. The high frequency seems to be relevant regarding the potential consequences of a hypothyroid state.

Background: Oral administration of ferrous-sulfate was reported to decrease intestinal absorption of mycophenolate mofetil (MMF) in healthy Japanese individuals by 90%. Methods: We examined the effect of a single oral dose of ferrous sulfate on steady-state mycophenolic acid pharmacokinetics in 10 iron-deficient (hypochromic red blood cells >2.5%), Caucasian, long-term kidney graft recipients using a randomized, open-label, crossover design. On days A and B, MMF (1,000 mg) was given orally at 8:00 am. On day C, MMF and ferrous sulfate (105 mg) were coadministered at 8:00 am. On day D, MMF was given at 8:00 am and ferrous sulfate was given orally 4 hours later. Results: The interindividual variability of the 12-hour area under the plasma mycophenolic acid concentration versus time curves (AUC 0–12 ) under control conditions was small (89.5 ± 27.8 and 87.6 ± 39.1 mg · h/L, respectively). Concomitant or subsequent administration of MMF and ferrous sulfate did not affect the bioavailabilty of MMF (AUC 0–12 , 91.9 ± 30.4 mg · h/L and 96.0 ± 31.7 mg · h/L). Conclusion: Oral therapy of iron deficiency using ferrous sulfate in long-term kidney graft recipients does not impede intestinal absorption of MMF; hence, exposure to this immunosuppressive agent is not reduced.

Anderson-Fabry disease is possibly underdiagnosed in patients with end-stage renal disease. Nationwide screening was therefore undertaken for Anderson-Fabry disease among dialysis patients in Austria. Screening for alpha-galactosidase A (AGAL) deficiency was performed by a blood spot test. In patients with a positive screening test, AGAL activity in leukocytes was determined. Individuals with decreased leukocyte AGAL activity were subjected to mutation testing in the GLA gene. Fifty (90.9%) of 55 Austrian hemodialysis centers participated in this study; 2480 dialysis patients (80.1% of the Austrian dialysis population) were screened. In 85 patients, the screening test was positive (85 of 2480, 3.42%; women, 3.32%; men, 3.50%). Among these 85 patients, 4 men (in 3 of whom Anderson-Fabry disease was already known before screening) had a severely decreased and 11 subjects had a borderline low AGAL activity. Genetic testing revealed mutations associated with Fabry disease in all four men with severely decreased AGAL activity resulting in a prevalence of 0.161% for the entire study population. A nationwide screening of dialysis patients permitted detection of a hitherto unknown man with Anderson-Fabry disease. The overall prevalence among dialysis patients was at least ten times higher as compared with recent registry data. Screening programs among patients with end-stage renal disease, especially men, should be put in place to identify families with Anderson-Fabry disease who probably may benefit from specific clinical care, and perhaps from enzyme replacement therapy. In dialysis patients, however, there is no evidence to support enzyme replacement therapy at present.

Controversy exists regarding the possible associations between a single nucleotide polymorphism of the transcobalamin II encoding gene (TCN2 776CG) and plasma levels of vitamin B(12), folate, or total homocysteine.In a cross-sectional study of 732 kidney allograft recipients, patients were categorized by TCN2 776CG genotype. In univariate and multivariate linear regression models that allowed the outcome variables vitamin B(12), folate, and total homocysteine plasma levels to follow a gamma distribution, we tested for possible associations of allelic variants of the TCN2 776CG gene and these three dependent variables.The allele frequency for TCN2 776CG was 0.46. Heterozygosity or homozygosity for TCN2 776CG was not associated with plasma levels of vitamin B(12) (776CG, P= 0.22; 776GG, P= 0.89), folate (776CG, P= 0.91; 776GG, P= 0.84), or total homocysteine (776CG, P= 0.11; 776GG, P= 0.33) even after adjustment for several possible confounders.We conclude from this largest study on the subject thus far that there are no associations between allelic variants of TCN2 776CG and plasma vitamin B(12), folate, or total homocysteine plasma levels in kidney transplant patients.

Atherosclerosis is a slow, complex process in which lipids, cholesterol, calcium and other substances are accumulating in the endothelium of the arterial wall. These deposits stimulate the artery wall cell to produce substances that lead to further thickening of the endothelium. In consequence the artery’s diameter shrinks, ultimately resulting in clinically evident vascular disease.

The association of variants of the gene encoding methionine synthase reductase ( MTRR ) with hyperhomocysteinemia, folate and Vitamin B 12 status in kidney graft recipients is unknown. We examined two mutations in MTRR in a cross-sectional study of 733 kidney graft recipients. The allele frequency of MTRR 66G was 0.55. 369 patients (50.3%) were heterozygous and 219 patients (29.9%) were homozygous for the mutation. None of the patients showed the 997C>G mutation. The allelic variants of MTRR 66A>G showed no significant association with total homocysteine (tHcy) levels, both in univariate analyses, and in a multivariate model controlling for age, gender, body mass index, renal function, time since transplantation, underlying kidney disease, as well as the MTHFR 677C>T/1298A>C genotypes. Similarly, no significant associations between the MTRR 66A>G genotypes and plasma folate or Vitamin B 12 levels were found. In conclusion, MTRR 66A>G has no major effect on tHcy, folate, or Vitamin B 12 plasma concentrations in kidney graft recipients.

Systemic mastocytosis (SM) is a clonal hematologic disease showing abnormal growth and accumulation of mast cells (MC) in visceral organs with or without skin involvement. The clinical course in SM is variable. In fact, indolent and aggressive variants have been described. In addition, SM patients may acquire an associated hematologic clonal non-MC lineage disease (AHNMD). In some cases, hematologic parameters are indicative of slowly progressing SM although the clinical course remains indolent over years. These cases have been referred to as smouldering SM. We report on a smouldering patient presenting with typical skin lesions, hypercellular marrow with focal MC aggregates, persistent leukocytosis (20,000-30,000/microl) with eosinophilia (5-10%), marked lymphadenopathy, and splenomegaly. The C-KIT mutation Asp-816-Val confirmed the diagnosis of SM. The clinical picture remained stable during an observation period of 10 years without signs of progression to an AHNMD or a high grade MC disease. These data show that some patients with SM can remain in a clinically indolent smouldering state over years even when presenting with marked eosinophilia and lymphadenopathy.

The effect of thiamine (vitamin B(1)) or riboflavin (vitamin B(2)) availability on fasting total homocysteine (tHcy) plasma levels in end-stage renal disease patients is unknown. A cross-sectional study was performed in a population of non-vitamin supplemented patients maintained on continuous ambulatory peritoneal dialysis. Red blood cell availability of thiamine (alpha-ETK) and of riboflavin (alpha-EGR), along with other predictors of tHcy plasma levels, was considered in the analysis. There was a linear association of alpha-EGR with tHcy plasma concentrations (P = 0.009), which was not observed for alpha-ETK. Among red blood cell vitamins, alpha-EGR was the only predictor of tHcy levels (P = 0.035), whereas alpha-ETK, red blood cell pyridoxal-5-phosphate supply (alpha-EGOT) and red blood cell folate levels had no effect. The risk for having a high tHcy plasma levels within the fourth quartile (plasma tHcy38.3 micromol/L) was increased by an alpha-EGRmedian (odds ratio, 4.706; 95% confidence interval, 1.124 to 19.704; P = 0.026). By way of contrast, alpha-ETK had no effect in these analyses. Independent predictors of tHcy plasma levels were serum albumin, alpha-EGR, red blood cell folate, and certain MTHFR genotypes. A logistic regression analysis showed that the MTHFR genotype is a predictor for having a tHcy plasma concentration within the fourth quartile. In summary, riboflavin availability, as measured by alpha-EGR, is a determinant of fasting tHcy plasma levels in peritoneal dialysis patients. This finding may have implications for tHcy lowering therapy in individuals with end-stage renal disease.

The influence of the genotype on the phenotypic expression of homocystinuria due to cystathionine beta-synthase (CBS) deficiency is frequently unclear. We therefore investigated the genotype and the phenotype of CBS deficiency in two Austrian families also considering genetic polymorphisms with a putative association with vascular disease (MTHFR 677C-->T, MTHFR 1298A-->C, F5 1691G-->A, F2 20210G-->A) and response to therapy. We identified the CBS 833T-->C/1058C-->T and CBS 828ins104/1358del134 compound heterozygous genotype in our index patients. Both patients showed mental retardation and ectopia lentis. CBS 833T-->C/1058C-->T was associated with severe vascular complications, which was not the case for CBS 828ins104/1358del134. The patient with CBS 828ins104/1358del134 was negative for F5 1691G-->A, F2 20210G-->A, MTHFR 677C-->T, and MTHFR 1298A-->C, while the patient with CBS 833T-->C/1058C-->T was heterozygous for MTHFR 1298A-->C. A combination therapy including pyridoxine, folic acid, hydroxycobalamin, and betaine failed to lower total homocysteine plasma levels below 50 mumol/L in both patients. In summary, our study demonstrates that the CBS 833C/1058T-MTHFR 1298AC genotype can be related to severe vascular disease, while the CBS 828ins104/1358del134-MTHFR 1298AA genotype presents with a somewhat milder clinical phenotype. Both genotypes do not allow for normalisation of total homocysteine plasma levels following vitamin therapy.

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 ( 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. © 2001 by the National Kidney Foundation, Inc.

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'.

The effectiveness of intravenous folinic acid or intravenous folic acid for the treatment of hyperhomocysteinemia of hemodialysis patients is unknown. In a randomized, controlled, double-blind trial, 66 hemodialysis patients were administered either 15 mg of folic acid or an equimolar amount (16.1 mg) of folinic acid intravenously three times weekly. Normalization of total homocysteine (tHcy) plasma levels after 4 weeks of treatment was achieved in 10 patients (30.3%) in the folic-acid group and 6 patients (18.2%; P: = 0.389) in the folinic-acid group (normalization at any time during the study period in 39.4% and 33.3% of the patients; P: = 0.798). The relative reduction in tHcy plasma levels at week 4 was 32.2% in the folic-acid group and 34.1% in the folinic-acid group. A high baseline tHcy plasma concentration (P: = 0.00001), methylenetetrahydrofolate reductase (MTHFR) 677TT/1298AA genotype (P: = 0.03540), and low red blood cell folate concentrations (P: = 0.02285) were associated with a better relative response to treatment. Normalization of tHcy plasma levels was dependent on a lower baseline tHcy level (P: = 0.01976), younger age (P: = 0.00896), and MTHFR 677TT/1298AA or 677CT/1298AC genotypes (P: = 0.00208 and P: = 0.02320, respectively). A 4-week course of intravenous folinic acid is not superior to intravenous folic acid in reducing elevated tHcy plasma levels in hemodialysis patients. The response to treatment is predicted by tHcy plasma level, red blood cell folate content, and MTHFR genotype.

An elevated total homocysteine (tHcy) plasma concentration is associated with increased morbidity and mortality due to cardiovascular disease in the general population and in patients with impaired renal function. The prevalence of hyperhomocysteinaemia (plasma levels above 15 micromol/l) in the general population is less than 5% and can be as high as 50% in patients with vascular disease. In patients with renal insufficiency, elevated tHcy plasma levels are detected in 50 - 100% of the patients. Total homocysteine plasma levels can be lowered or normalised by folic acid and/or vitamin B(6) and vitamin B(12) supplementation. In patients with advanced chronic renal insufficiency or end-stage renal disease, hyperhomocysteinaemia is partially resistant to folic acid or vitamin therapy. However, higher tHcy plasma levels may also reflect tissue damage and the increase in Hcy after an acute incident such as stroke or myocardial infarction may be necessary for tissue repair mechanisms. This implies, that lowering tHcy may even be harmful to some patients. Currently, prospective studies are underway to clarify whether folate supplementation, with or without additional other vitamins, improves cardiovascular disease morbidity and mortality in the general population, as well as in renal failure patients. While population-wide screening for and treatment of hyperhomocysteinaemia is generally not recommended, treatment of high risk patients may be considered.

The effect of 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C--T and 1298A--C on total homocysteine (tHcy), folate and vitamin B(12) levels was investigated in 733 kidney graft recipients. The six major genotype combinations were used as grouping variables, and age, gender, BMI, serum creatinine, and creatinine clearance and ln-folate, ln-vitamin B(12), or logarithmus naturalis tHcy (ln-tHcy) were used as covariates in three ANCOVA and multiple stepwise linear regression models. Hyperhomocysteinemia was present in 49.7% of the patients. The allele frequency of MTHFR 677T and 1298C was 0.319 and 0.326. MTHFR genotype and all other variables were significant predictors of ln-tHcy (higher tHcy plasma levels for MTHFR 677TT/1298AA versus all other five genotype groups: P0. 05). BMI, creatinine clearance, ln-tHcy, and MTHFR genotype influenced ln-folate (lower folate levels for MTHFR 677TT/1298AA versus all other genotype groups: P0.05). Creatinine clearance and ln-tHcy were the only predictors of ln-vitamin B(12) levels. In a prespecified subgroup analysis (n = 496), the MTHFR genotype also influenced tHcy levels and compound heterozygous patients had significantly lower folate levels as compared with MTHFR 677CC/1298AA and 677CC/1298CC. This study shows that the MTHFR 677TT/1298AA and 677CT/1298AC genotypes are significant predictors of tHcy and folate plasma levels.

An elevated total homocysteine plasma concentration is associated with an increased morbidity and mortality due to cardiovascular disease in the general population, in patients with renal failure and in recipients of kidney or heart transplants. The fasting or post-methionine loading plasma concentration of total homocysteine is elevated in 50-60% of renal transplant recipients with stable graft function and in the majority of heart transplant recipients. Fasting and post-methionine loading hyperhomocysteinemia can be normalized in virtually all renal transplant patients by a combination of folic acid (5 mg/d), vitamin B6 (50 mg/d) and vitamin B12 (0.4 mg/d). In individuals without renal failure much lower doses of folate and vitamin B12 are able to correct hyperhomocysteinemia. Currently, prospective studies are under way to clarify whether folate and vitamin therapy improves cardiovascular disease morbidity and mortality in the general population and in organ transplant recipients. While population wide screening for and treatment of hyperhomocysteinemia is generally not recommended, treatment of high risk patients, including renal failure patients and kidney and heart transplant recipients, can be considered but still represents an experimental therapy.

Recent genetic studies have led to the characterization of molecular determinants contributing to the pathogenesis of hyperhomocysteinemia. In this article we summarize the current insights into the molecular genetics of severe, moderate and mild hyperhomocysteinemia. We will consider deficiencies of the trans-sulfuration enzyme cystathionine β-synthase (gene symbol: CBS), and the disturbances of the remethylation enzymes 5,10-methylenetetrahydrofolate reductase (gene symbol: MTHFR), methionine synthase (gene symbol: MTR), and the recently identified methionine synthase reductase (gene symbol: MTRR). Furthermore, we will focus on clinically important genetic polymorphisms which are highly prevalent and thus of potential general interest.

Hyperhomocysteinemia is a cardiovascular disease risk factor in the general population and in patients with renal failure. This article summarizes the results of recent total homocysteine-lowering intervention studies in individuals without renal failure and in patients with renal failure. Although almost all of the published studies mainly focused on the total homocysteine-lowering effect and not on cardiovascular disease outcomes of folic acid and vitamin therapy, recent work has gained important insights into this area of developing concern.

The present study analyzes the efficiency of a combination of four immunoglobulin heavy chain (IgH) gene polymerase chain reaction (PCR) primer systems and a multiplex T-cell receptor gamma chain (TRG) gene PCR for detection of clonality in 409 samples (234 paraffin sections, 175 bone marrow aspirates) of different lymphomas. Using the four IgH PCR systems together, clonality was detected in all samples of B-cell chronic lymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and Burkitt-like B-cell lymphomas. Clonality was detected in all bone marrow aspirates with lymphoplasmacytoid immunocytoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and unclassifiable low-grade B-cell lymphomas. The combined IgH gene PCR approach allowed clonality detection in 78.2% of myelomas, 75% of Burkitt lymphomas, 74.4% of diffuse large B-cell lymphomas, 68.7% of follicular center lymphomas, 50% of posttransplant lymphomas, 28.6% of anaplastic large cell lymphomas, 29% of T-cell lymphomas, and 18.8% of Hodgkin diseases. The combination of the four IgH gene primer systems with the multiplex TRG gene PCR allowed detection of clonality in 84.2% of B-cell neoplasms, 92.1% of T-cell non-Hodgkin lymphomas, and 18.8% of Hodgkin diseases, which was much more efficient than single PCR protocols.

Mast cells (MC) are multipotent hemopoietic effector cells producing diverse mediators like histamine, heparin, or tissue type plasminogen activator. We report a 75-year-old male patient with myelodysplastic syndrome (MDS) of recent onset (3 months' history) associated with a massive leukemic spread of immature tryptase+ MC (tentative term: myelomastocytic leukemia). The patient presented with pancytopenia, bleeding, hypofibrinogenemia, and an increased cellular tryptase level. Moreover, an excessive elevation of plasmin-antiplasmin complexes (9,200 ng/ml; normal range: 10-150), an elevated D-dimer, and an increase in thrombin-antithrombin III complexes were found. The identity of the circulating MC was confirmed by immunophenotyping (CD117/c-kit+, CD123/IL-3R alpha-, CD11b/C3biR-), biochemical analysis (cellular ratio [ng:ng] of tryptase to histamine >1), and electron microscopy. Bone marrow (bm) examination showed trilineage dysplasia (17% blasts), 30% diffusely scattered MC, and a complex karyotype. No dense, compact MC infiltrates (mastocytosis) were detectable in bm sections. Despite hyperfibrinolysis and mediator syndrome (flushing, headache), the patient received remission induction polychemotherapy (DAV) followed by two cycles of consolidation with intermediate dose ARA-C (2 x 1 g/m2/day on days 1, 3, and 5). He entered complete remission after the first chemotherapy cycle without evidence of recurring MDS. Moreover, in response to chemotherapy, the hyperfibrinolysis and mediator syndrome resolved, and the circulating c-kit+ MC disappeared. We suggest consideration of polychemotherapy as a therapeutic option in patients with high-risk MDS of recent onset, even in the case of MC lineage involvement.

Background: Mutations in the carboxy termini of the beta subunit (hβENaC) and the gamma subunit (hγENaC) 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 hβENaC and hγENaC 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 hβENaC and hγENaC 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 hβENaC and hγENaC genes in Austrian patients with ESRD maintained on chronic hemodialysis treatment. Thus, mutations in these genes are unlikely to be associated with ESRD.

Mutation 677C to T in the methylenetetrahydrofolate reductase gene aggravates hyperhomocysteinemia in hemodialysis patients. Hyperhomocysteinemia is frequent in hemodialysis patients and represents an independent risk factor for vascular disease in these patients. Elevated total homocysteine (tHcy) plasma levels can result from defective remethyla-tion of Hcy to methionine due to decreased activity of the enzyme methylenetetrahydrofolate reductase (MTHFR). A genetic aberration in the MTHFR gene (677C to T substitution) has been shown to result in reduced MTHFR activity. We tested the hypothesis that elevation of tHcy plasma levels in hemodialysis patients is influenced by the 677C to T mutation of the MTHFR gene and examined the relation of the genotype with tHcy, folate and vitamin B 12 plasma levels in these patients. The allelic frequency of the MTHFR mutation was evaluated in 203 patients maintained on chronic hemodialysis treatment. Total Hcy, folate, vitamin B 12 levels and the MTHFR mutation were analyzed in 69 of the 203 patients and in 69 age- and sex-matched healthy control subjects. The allelic frequency of the 677C to T transition in the MTHFR gene in hemodialysis patients was 34.7% versus 35.5% in healthy controls. Of 203 patients 26 (12.8%) were homozygous for the mutation (+/+) versus 10.2% in healthy subjects. The heterozygous (+/−) genotype was identified in 43.8% of patients versus 50.7% in controls. The mean tHcy level in hemodialysis patients was 28.7 ± 11.0 µ uunol/liter versus 10.0 ± 3.0 µ mol/liter in control subjects. The mean tHcy levels were 36.4 ± 13.4 µ mol/liter in (+/+) patients and 12.2 ± 4.5 /mol/liter in (+/+) controls, 28.7 ± 10.8 µ mol/liter in (+/−) patients and 9.9 ± 2.7 µ mol/liter in (+/−) controls and 25.4 ± 8.5 µ mol/liter in (−/−) hemodialysis patients versus 9.7 ± 2.8 µ mol/liter in (−/−) controls. There was no significant difference of folate and vitamin B 12 concentrations in patients and controls with different MTHFR genotypes. Analysis of covariance including age, gender, folate concentrations, vitamin B 12 levels, albumin and creatinine as covariables revealed a significant influence of the (+/+) genotype, albumin and folate status on tHcy levels in hemodialysis patients. Together, our data demonstrate that the extent of hyperhomocysteinemia in hemodialysis patients is not only the result of uremia or folate status, but is also genetically determined by the (+/+) MTHFR genotype. The presence of the 677C to T mutation in the MTHFR gene does not appear to represent a risk factor for development of end-stage renal disease.

In this study, the authors evaluated a closed tube total hematology analysis system, Sysmex HS-430 (HS), which consists of two automated hematology analyzers, Sysmex NE-8000, the reticulocyte analyzer, Sysmex R-3000, and a slide preparation unit integrated in an automated sample transport system (TOA Medical Electronics, Kobe, Japan). The suitability of the system was compared with a conventional hematology system (CS) consisting of two single standing Coulter STKS analyzers (Hialeah, FL), one automated reticulocyte counter Sysmex R-1000, and the manual blood smear preparation. Evaluation was performed following the concept suggested by the Austrian-German societies for Clinical Chemistry and Laboratory Medicine that includes the evaluation of personnel supply. Eight consecutive series with a total of 4,896 samples were analyzed on both systems. Evaluation revealed that the analysis time per series was 238 minutes on the HS-430 and 359 minutes on the CS. The mean analyzer down times because of technical reasons were 36 minutes on the HS and 32 minutes on the CS. The down time because of "nontechnical" reasons was 31 minutes on the HS-430, but 173 minutes on the CS, which was mainly because of discontinuous sample loading of analyzers of the CS. The average direct technical time effort for complete blood cell count (CBC) analyses, reticulocyte counts, and blood smear preparations per series was 23 minutes on the HS and 245 minutes on the CS. In summary, these data show that an automated system like the HS-430 saves 222 minutes of manual activities arising in a large routine hematology laboratory with a mean throughout of 612 samples per day. Furthermore, the system improves intralaboratory turnaround times, avoids human errors by automated sample identification, and guarantees more safety for laboratory staff by minimizing the contact with potential biohazards.

We have compared the kinetics of minimal residual disease (MRD) by simultaneous polymerase chain reaction (PCR) monitoring with oligonucleotides for the immunoglobulin heavy chain (IgH) complementarity-determining region 3 (CDR3) and the T-cell receptor gamma chain gene (TCR gamma), as well as clone-specific CDR3 sequences in adult patients (aged 17-51 years) with acute lymphoblastic leukemia (ALL) who entered a complete hematological remission (CR) after chemotherapy with the German multicenter ALL (GMALL) protocol. The sensitivities were one in 10(2-3) for the CDR3- and TCR gamma-PCR and one in 10(5-6) for a two-step, seminested CDR3/clone-specific PCR. At diagnosis, 7/7 patients were CDR3 positive and four were TCR gamma positive in their bone marrow (BM). At the end of induction therapy (after 2 months) 4/6 tested positive for CDR3, 2/6 for TCR gamma, and 5/6 for clone-specific rearrangements. At the end of consolidation treatment (after 7 months) only 1/7 remained positive for CDR3, 2/7 for TCR gamma, and 5/7 for clone-specific rearrangements. After an observation period of 18-36 months, 4/7 patients were still in CR and all were PCR negative by the clone-specific method during or after maintenance therapy. Two patients died in leukemic relapse; one patient relapsed but is still alive. All three of these patients remained PCR positive throughout the course of their disease. Clonal evolution in the IgH locus was found in one of these patients. We conclude that the molecular response to chemotherapy in adult B-lineage ALL is slow, even in patients without risk factors other than age. As in childhood ALL, most patients with long-term CR convert to PCR negativity approximately 18 months after the start of chemotherapy. The data also suggest the existence of early clone-specific PCR negativity in a small proportion of long-term survivors. The predictive value of this observation will now have to be confirmed in a larger study.

We report on a patient with cutaneous T-cell lymphoma (CTCL) of long-standing duration. Phenotypic analysis of his peripheral blood mononuclear cells revealed an increased CD4 + T-helper subset and a decreased CD8 + cytotoxic T-cell population. Eighty-three to ninety-three percent of the patient's CD4 + T cells in the peripheral blood and 70% of the CD + T cells in the lesional skin lacked surface expression of the TCR/CD3 complex and showed a clonal rearrangement pattern of the TCR gammachain gene (V11-J1/J2). The lack in TCR surface expression correlated with defective assembly of the TCR beta-chain. Although mRNA for the TCR constant region β1 was found in the patient's purified CD4 + TCR - CD3 - T cells, no intracytoplasmic TCR beta protein was detectable. In contrast, the patient's purified CD4 + TCR - CD3 - T cells not only expressed mRNA specific for the TCR alpha-chain and for all CD3 chains, but intracytoplasmic TCR alpha and CD3 epsilon proteins could also be found. The lack of TCR beta protein clearly explain the defective surface expression of the TCR/CD3 complex in the patient's malignant T cells.

We report the results of consecutive tests in nine BCR-ABL-positive ALL patients by one-step and two-step (nested primer) reverse transcriptase-polymerase chain reaction (RT-PCR). Six patients could be tested in complete haematological remission (CHR). One patient remained one-step positive; four patients became one-step negative, but remained two-step positive; only one patient became two-step negative. In five patients the haematological relapse was preceded by one-step positivity by 1.5-5 weeks. In two patients who received autologous BMT in CHR, BCR-ABL was still detectable by two-step PCR, whereas allogeneic BMT was able to transiently reduce BCR-ABL below the two-step detection level. Our results show that one-step combined with two-step RT-PCR analysis gives valuable information about the efficacy of treatment and the dynamics of the leukaemic clone.

TO THE EDITOR: Keay et al.1 recently reported detection of bacterial 16s rRNA in about 60% of needle biopsy samples obtained from patients with localized adenocarcinoma of the prostate using a two-step polymerase chain reaction (PCR) protocol.2,3 Application of the same PCR protocol allowed detection of bacterial DNA in 77% of transperineal prostate biopsies of patients with chronic prostatitis.4 The authors therefore conclude that the presence of bacterial 16s rRNA in prostate tissue is not specific for chronic prostatitis and localized prostate cancer. PCR analysis is a very sensitive method that allows for the detection of a single DNA target in a sample. In the past, the extremely high sensitive two-step PCR has been introduced into routine molecular diagnostics for therapeutic monitoring of patients with minimal residual leukemia.5 In contrast, two-step PCR protocols are usually not applied in microbiologic diagnostics because of the questionable clinical relevance of a positive result that may have arisen from as few as a single bacterial DNA molecule. Because of the exquisite sensitivity of PCR, precautions have to be taken to ensure that positivity indeed indicates the presence of bacterial DNA in the tissue sample and that the positive signal does not result from exogenous sources.6 Moreover, to avoid false negativity, a DNA control has to be amplified to exclude the possibility that a negative result is due to the lack of DNA in the samples (ie, failure of the DNA isolation procedure) or the presence of PCR inhibitors. Among the studies that have used the two-step PCR protocol published by Domingue et al.2 and Keay et al.,3 controversial detection rates have been reported with respect to bacterial DNA detected in samples of patients with interstitial cystitis and in those of healthy controls.2,3 Beside the possibility of false positivity resulting from exogenous contamination of the specimens during manipulation of the cystoscope,3 these differences may also be due to false negativity of PCR because neither of the investigators included a DNA control in the PCR protocol to confirm the presence of amplifiable DNA in the samples. Because one-step PCR protocols represent a useful tool to investigate the prevalence of bacteria that fail to grow in culture, we highly recommend inclusion of a DNA control (ie, amplification of the human beta-globin gene)7 in all PCR protocols to avoid false negativity of PCR in tissue biopsy samples.

Peritoneal elimination of homocysteine moieties in continuous ambulatory peritoneal dialysis patients. Background The amount of total homocysteine eliminated by peritoneal dialysis and its relationship to peritoneal transport characteristics in continuous ambulatory peritoneal dialysis (CAPD) patients are unknown. Methods The influence of total homocysteine, folate, and vitamin B 12 plasma concentrations, serum albumin levels, age, sex, dialysate to plasma ratio (D/P) creatinine, D/D 0 glucose, D/P albumin, dialysate effluent volume, and effluent albumin on the daily peritoneal excretion of total homocysteine was investigated in 39 CAPD patients. The relationship of D/P creatinine to D/P total homocysteine, D/P free homocysteine, and D/P protein-bound homocysteine was analyzed additionally in a subgroup of 25 patients. Results We observed a significant influence of plasma total homocysteine concentrations ( P = 0.0001) of the daily dialysate effluent volume ( P = 0.0221) and of the D/P creatinine ( P = 0.0132) on peritoneal elimination of total homocysteine. The daily peritoneal excretion of total homocysteine was 38.94 ± 20.82 μmol (5.27 ± 2.81mg). There was a positive linear association of the daily total homocysteine elimination with plasma total homocysteine concentrations ( P = 0.0001). A significant linear correlation was observed between D/P creatinine and D/P total homocysteine ( P = 0.0001), D/P free homocysteine ( P = 0.0001), as well as D/P protein-bound homocysteine ( P = 0.0001). Conclusions The peritoneal elimination of total homocysteine primarily depends on the plasma total homocysteine concentration. Elevated total homocysteine plasma levels cannot be reduced efficiently by peritoneal dialysis.

Genetic determinants of the homocysteine level. Elevated total homocysteine (tHcy) plasma concentrations indicate folate and/or vitamin B12 deficiency and are associated with cardiovascular disease and neural tube defects. Evidence has accumulated that folate-, vitamin B12-, and Hcy-metabolism are under genetic control. Because Hcy metabolism is impaired in renal failure, MTHFR 677 C>T, GCP2 1561C>T, RFC1 80G>A, and TCN2 776G>C may further aggravate hyperhomocysteinemia in these patients. The most consistent effect on tHcy plasma concentrations is observed for 677C>T of MTHFR, whereas GCP2, RFC1, and TCN2 polymorphisms show no major effect on tHcy concentrations. Much is yet to be learned about the impact of genetic variants on tHcy levels, human diseases, the genetic-nutrient interactions, as well as the pharmacogenetic consequences in Hcy and vitamin metabolism.

Iron overload in kidney transplants: Prospective analysis of biochemical and genetic markers. Background The prevalence of iron overload and the influence of mutations in the HFE and TRF2 gene on biochemical markers of iron overload among renal transplant patients is unknown. Methods Serum iron, ferritin, transferrin saturation (TSAT), and liver function parameters were analyzed in a cohort of 438 renal transplants. In patients with iron overload, the time course of biochemical markers of iron status as well as the influence of iron loading mutations was investigated during a time period of 5 years. Results Of 438 renal transplant patients 41 (9.4%) presented with an iron loading phenotype (TSAT above 40% and/or ferritin above 800ng/mL). Mutations in the HFE gene were present in 12 of 33 (36.3%) patients with iron overload. Among these one patient was homozygous for HFE C282Y, and two patients were compound heterozygous for HFE C282Y/H63D. No individual tested positive for nine other mutations in HFE as well as the TRF2 Y250X mutation. Over time we observed a decrease of mean iron and ferritin levels, and of mean TSAT in our study sample. In patients with mutations in HFE this decrease was less pronounced as compared to patients without mutations. We found an independent positive association between the presence of mutations in HFE and serum alanine-aminotransferase levels at follow-up ( P = 0.003). Conclusion Our study demonstrates that iron overload is frequently present in renal transplant patients and shows a continuous decrease over time. This decrease is possibly impaired by the HFE C282Y and HFE H63D mutations. Furthermore, mutations in HFE may influence liver function as reflected by increased alanine-aminotransferase concentrations.

Inherited disorders of iron metabolism. Recent molecular studies have resulted in the identification of genetic alterations underlying hereditary disorders of iron metabolism. One example is the discovery of the HFE gene that is mutated in patients suffering from hereditary hemochromatosis. This autosomal recessive disorder has an estimated carrier frequency that varies between 0.07 and 0.13, thus representing one of the most common genetically determined metabolic disorders. The identification of the hemochromatosis mutations has encouraged efforts to investigate other conditions with iron overload for a putative interaction with these genetic variants. Few data are already available suggesting, for example, that iron overload in patients with sporadic porphyria cutanea tarda is associated with mutations in the hereditary hemochromatosis gene. However, it is obvious that disorders of iron metabolism have a multifactorial pathogenesis, including environmental and genetic factors. Thus, many questions remain to be answered about whether a genetic predisposition exists for development of various iron-loading or iron-deficiency phenotypes. This review focuses on the most recent advances in the field of hereditary disorders of iron metabolism and discusses their potential implications for nephrologists.

Background. Vascular access thrombosis represents a serious problem in haemodialysis patients. Therefore identification of relevant thrombotic risk factors is clinically valuable. Resistance to activated protein C (APC) was recently identified as a new thrombophilic defect which is caused by a single point mutation in the factor V gene. Whether this mutation predisposes to vascular access thrombosis is unknown. Methods. The presence of factor V Leiden (mutation at nucleotide position 1691 of the factor V gene) was determined by polymerase chain reaction (PCR) analysis in 152 haemodialysis patients from all three haemodialysis units of the University Hospital of Vienna. In 61 patients (54 without mutation, 7 with heterozygous mutation) resistance to APC was evaluated. Onehundredseven individuals without renal failure (57 negative for factor V Leiden, 50 heterozygous subjects) served as controls. Haemodialysis patients with heterozygous factor V Leiden mutation were carefully investigated for thrombotic complications of vascular access, other thromboembolic events and additional putative thromboembolic risk factors. Results. Seven of 152 (4.6%) patients were heterozygous carriers of factor V Leiden. The mean APC resistance ratio in heterozygous dialysis patients was 2.31 ; in the 50 heterozygous controls the ratio was 2.02. The mean APC ratio in haemodialysis patients without mutation was 3.53 in contrast to 2.95 in the control group. Not one of the seven heterozygous haemodialysis patients suffered from vascular access thrombosis of inexplicable origin. Three patients remained totally free of access thrombosis from onset of haemodialysis treatment. In four of seven patients nine events of thrombosis of the vascular access occurred, but were due to anatomical stenosis in each case. In six permanent central venous catheters no episode of occlusion or reduced blood flow requiring thrombolytic therapy was observed. Family history with regard to thrombotic events was negative in all seven patients. No thromboembolic complication occurred during 13 periods of immobilization, in the course of six pregnancies and during oral contraception. Conclusions. The heterozygous carrier status for factor V Leiden does not appear to represent a risk factor for vascular access thrombosis in haemodialysis patients. This is possibly due to the fact that the functional APC activity is high and in heterozygous haemodialysis patients APC resistance ratios are very close to the normal range. However, it cannot be excluded that a homozygous factor V mutation represents an increased risk for shunt thrombosis. Therefore patients suffering from repeated and/or inexplicable shunt thrombosis should be tested for the factor V mutation to evaluate the effect of a homozygous mutation.

The effect of the combined 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T and 1298A-->C genotype on total homocysteine (tHcy), folate, and vitamin B(12) plasma levels was investigated in 983 subjects, including 415 hemodialysis patients, 179 peritoneal dialysis patients, and 389 healthy individuals. Mean tHcy plasma concentrations were 27.2 +/- 15.8 micromol/L in hemodialysis patients, 25.4 +/- 19.1 micromol/L in peritoneal dialysis patients, and 8.9 +/- 3.5 micromol/L in healthy individuals. Hyperhomocysteinemia (tHcy > 15 micromol/L) was detected in 81.6% of patients and 2.6% of controls. Multiple stepwise regression analysis showed that the MTHFR 677C-->T/1298A-->C genotype (CC/AA, CC/AC, CC/CC, CT/AA, CT/AC, TT/AA), vitamin use, age, folate and vitamin B(12) plasma level were significant predictors of tHcy plasma levels. Analysis of variance showed that this effect of MTHFR genotypes on tHcy level was caused by significantly greater tHcy levels in 677TT/1298AA hemodialysis and peritoneal dialysis patients versus other genotypes. Compound heterozygous controls (677CT/1298AC genotype) had significantly greater tHcy levels compared with 677CC/1298AA controls. There was no major effect of MTHFR polymorphisms on folate and vitamin B(12) plasma concentrations. This study shows that the MTHFR 677TT/1298AA genotype, but not the 677CT/1298AC genotype, is a significant predictor of tHcy plasma levels in dialysis patients.