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Eur J Clin Invest 2011; 41 (6): 579–583 Abstract Background Over the years, the demonstration and confirmation of cell-free DNA in the circulation has increasingly been recognized as a valuable diagnostic tool. Likewise, it has been known for some time that DNA structures that are targeted by auto-antibodies play a central role in systemic lupus erythematosis (SLE) and that DNA-antibody complexes in the circulation are one of the hallmarks of SLE. Investigating whether and to what degree fluctuations in free plasma DNA levels in patients with SLE might correspond to disease severity was therefore the goal of this investigation. Methods Blood from 13 patients with SLE and from 13 healthy controls was taken and analysed for the presence of anti-dsDNA, anti-ssDNA, anti-nucleosome, anti-histone antibodies as well as for cell-free DNA concentrations. For each patient, the SLE disease activity index (SLEDAI) was calculated. Results As demonstrated herein, compared to healthy subjects, cell-free DNA plasma levels in patients with SLE were significantly increased and so were anti-dsDNA, anti-ssDNA, anti-histone and anti-nucleosome antibodies. Furthermore, a statistically significant correlation was noted between cell-free DNA and anti-histone antibodies in patients with SLE. However, no correlation was noted between disease activity and anti-dsDNA, anti-ssDNA and anti-nucleosome antibody concentrations. Surprisingly, and more important in the context of this study, there was no correlation between cell-free DNA levels and SLEDAI scores. Conclusions The presented data seem to exclude measuring free plasma DNA as an inexpensive, simple and quick tool to assess disease activity in patients with SLE. Further studies on a larger patient population would be needed to confirm our results.

The KIT mutation D816V is associated with autonomous growth of mast cells (MC) and is detectable in most patients with systemic mastocytosis (SM), including cases with associated hematologic non‐MC‐lineage disease (AHNMD). Recently, KIT D816V was reported to be expressed in patients with acute myeloid leukemia (AML). However, it was not clarified whether these patients have co‐existing occult SM. We investigated neoplastic cells in 101 patients with AML for expression of KIT D816V. In 7/101 patients (6.9%), KIT D816V was detectable. After a thorough histologic, molecular, and biochemical analysis, all 7 cases were found to have an associated SM, leading to the final diagnosis SM‐AML. Microdissected tryptase+ MC displayed KIT D816V in all patients tested, whereas CD34+ blasts exhibited KIT D816V in only 2/4 patients. In one AML patient, SM without KIT D816V was detected. In all other patients, no associated SM was found, and leukemic blasts were negative for KIT D816V. In summary, our data show that KIT D816V in AML is highly associated with co‐existing SM (SM‐AML). Moreover, our data show that AML blasts may lack this transforming target‐mutant, which may be important when considering the use of KIT D816V‐targeting drugs for treatment of patients with KIT D816V‐positive AML.

Although a classification for mastocytosis and diagnostic criteria are available, there remains a need to define standards for the application of diagnostic tests, clinical evaluations, and treatment responses. To address these demands, leading experts discussed current issues and standards in mastocytosis in a Working Conference. The present article provides the resulting outcome with consensus statements, which focus on the appropriate application of clinical and laboratory tests, patient selection for interventional therapy, and the selection of appropriate drugs. In addition, treatment response criteria for the various clinical conditions, disease-specific symptoms, and specific pathologies are provided. Resulting recommendations and algorithms should greatly facilitate the management of patients with mastocytosis in clinical practice, selection of patients for therapies, and the conduct of clinical trials.

Appropriate measurement of the glomerular filtration rate (GFR) is important for the assessment of renal function. This paper reviews the methods used to assess GFR in clinical trials of enzyme replacement therapy (ERT) in patients with Fabry disease, which include inulin clearance, 24-hour creatinine clearance, chromium ethylene diamine tetraacetate ( 5 1 Cr-EDTA) clearance and cystatin C concentrations. GFR has also been estimated using calculations based on creatinine clearance (the Cockcroft-Gault formula) and the Modification of Diet in Renal Disease (MDRD) equation. Analysis of the results of these studies shows that there are striking discrepancies between estimated and measured GFR. For example, the MDRD equation overestimates GFR in patients with Fabry disease who have normal renal function. In addition, cystatin C has been shown to be of limited use for measuring renal function during ERT, because it is influenced by other factors such as age, gender and weight. Conclusion: The use of exact methods, such as inulin clearance, 1 2 4 I-iothalamate, 9 9 mTc-DTPA, 5 1 Cr-EDTA and iohexol, appears to be mandatory for a robust evaluation of the effects of ERT on GFR in patients with Fabry disease.

Background Recently, a new mutation of the 5,10-methylenetetrahydrofolate reductase (MTHFR) encoding gene was first described (1793G > A). Only few reports have studied the prevalence of this polymorphism, especially in combination with other MTHFR mutations (677C > T, 1298A > C). Methods We cross-sectionally identified the novel MTHFR 1793G > A polymorphism in 730 kidney transplant recipients. MTHFR 677C > T and 1298A > C were also assessed and the frequency of each was described individually as well as in cross-tabulation with the other MTHFR genotypes. The expected number of patients for each MTHFR genotype combination was calculated and contrasted with the observed numbers. Fisher's exact test was used for statistical inference. Results The allelic frequency of MTHFR 1793G > A was 0·052. Seventy-two patients (9·9%) were heterozygous and two patients (0·3%) were homozygous. From the cross-tabulations, we identified 53 patients (expected: 33·6) with the MTHFR 1298AC/1793GA genotype and 17 patients (expected: 6·7) with the MTHFR 1298CC/1793GA genotype. Furthermore, we found two patients with double homozygosity for MTHFR 1793G > A and MTHFR 1298A > C (MTHFR 1793AA/1298CC genotype). The frequencies of these genotype combinations were substantially larger than could be expected (P A and MTHFR 1298A > C genotypes, possibly owing to a mutually stabilizing effect on MTHFR enzyme activity.

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

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 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-M0 (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 (

Background The pathophysiology of hypertensive crises is poorly understood. To date, no information is available about genetic determinants underlying the individual risk for development of hypertensive urgencies or emergencies. Recently, mutations in the beta subunit (h beta ENaC) and the gamma subunit (h gamma ENaC) of the human epithelial sodium channel (hENaC) have been shown to result in excessive elevation of blood pressure in patients with Liddle's syndrome. Methods Using polymerase chain reaction and direct sequencing of amplification products we have screened 90 consecutive out-patients with hypertensive urgency or hypertensive emergency for the presence of mutations in the carboxy terminus of these genes. Furthermore, serum potassium concentrations were determined in all 90 patients, and serum aldosterone levels and plasma renin activity were measured in a subset of 34 patients. Results Among 71 patients with hypertensive urgency (78.9%) and 19 patients with hypertensive emergency (21.1%) not one individual showed a mutation in genomic DNA extending from codon 532 to codon 637 of h beta ENaC and from codon 525 to codon 651 of h gamma ENaC. Twelve of 90 patients showed mild hypokalaemia (13.3%), 16 of 34 patients had a plasma renin activity below the lower normal range (47.1%) and one of 34 patients had a low serum aldosterone concentration (2.9%). Conclusions The present study clearly demonstrates the absence of mutations in the carboxy terminus of the h beta ENaC and h gamma ENaC gene of hENaC in an Austrian cohort of 90 patients suffering from hypertensive crisis.

We describe a multiplex polymerase chain reaction (PCR) method suitable for the detection of all T-cell receptor (TCR) gamma-chain gene rearrangements in patients with lymphoproliferative diseases. 40 patients with various lymphoproliferative disorders and 40 healthy individuals were tested. Clonal TCR gamma rearrangements were identified in all patients with malignant disorders, and in one of 10 cases with established reactive lymphocytosis but not in normal controls. In all individuals testing positive, the patient's specific V and J segment involved in the rearrangement could be determined by simply splitting the multiplex primer mix. Our data show that the multiplex PCR technique enables rapid, simple and sensitive screening for clonal TCR gamma chain gene rearrangements.