Mutational landscape reflects the biological continuum of plasma cell dyscrasias.

We subjected 90 patients covering a biological spectrum of plasma cell dyscrasias (monoclonal gammopathy of undetermined significance (MGUS), amyloid light-chain (AL) amyloidosis and multiple myeloma) to next-generation sequencing (NGS) gene panel analysis on unsorted bone marrow. A total of 64 different mutations in 8 genes were identified in this cohort. NRAS (28.1%), KRAS (21.3%), TP53 (19.5%), BRAF (19.1%) and CCND1 (8.9%) were the most commonly mutated genes in all patients. Patients with non-myeloma plasma cell dyscrasias showed a significantly lower mutational load than myeloma patients (0.91±0.30 vs 2.07±0.29 mutations per case, P=0.008). KRAS and NRAS exon 3 mutations were significantly associated with the myeloma cohort compared with non-myeloma plasma cell dyscrasias (odds ratio (OR) 9.87, 95% confidence interval (CI) 1.07-90.72, P=0.043 and OR 7.03, 95% CI 1.49-33.26, P=0.014). NRAS exon 3 and TP53 exon 6 mutations were significantly associated with del17p cytogenetics (OR 0.12, 95% CI 0.02-0.87, P=0.036 and OR 0.05, 95% CI 0.01-0.54, P=0.013). Our data show that the mutational landscape reflects the biological continuum of plasma cell dyscrasias from a low-complexity mutational pattern in MGUS and AL amyloidosis to a high-complexity pattern in multiple myeloma. Our targeted NGS approach allows resource-efficient, sensitive and scalable mutation analysis for prognostic, predictive or therapeutic purposes.

A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Controlling the release and production of renin.

The renin-angiotensin system (RAS) plays a pivotal role for a variety of cardiovascular functions. The diversity of renin actions is reflected by its complex control. The major stimulus for the release of renin from the vesicles in juxtaglomerular cells is determined by stretch, as induced by changes in arterial pressure. The production of renin underlies a vastly complex control network, which takes place at different levels, such as transcription and translation. With regard to transcription, important regions for binding transcription factors have been identified several years ago, but the conservation of nucleotide sequences throughout different species suggests that there might be additional binding regions of importance. At the post-transcriptional level, the modulation of renin mRNA stability is seems pivotal. The half-life of renin mRNA appears to be controlled by the interaction between several regulatory proteins, most of which are well known in other systems. Moreover, in addition to the modulation of mRNA stability, the translation efficiency seems to play a key role in determining the amount of renin to be produced.

Endocrine-disrupting nonylphenols--ultra-trace analysis and time-dependent trend in mussels from the German bight.

A very sensitive and efficient analytical procedure is presented for the determination of 4-nonylphenols (NP) in blue mussels by use of off-line coupling of high-performance liquid chromatography (HPLC) and gas chromatography with mass spectrometric detection (GC-MS). Combined steam distillation and solvent extraction were used to extract the analytes from the mussel samples. Before quantification by GC-MS the raw extracts were purified by normal-phase HPLC. 4-n-Nonylphenol was used as internal standard. The detection limit was 15 ng NP absolute, calculated from the blank value. The method was applied to the determination of NP in blue mussel samples from the German North Sea sampled over a period of 10 years. Collection, homogenization, and storage of the mussels were performed according to the Standard Operating Procedures of the German Environmental Specimen Bank since 1985. The total NP concentrations in the mussels decreased significantly from 1985 (4 microgram kg (-1)) to 1995 (1.1 microgram kg (-1)).

Second German consensus on immunogenetic donor search for allotransplantation of hematopoietic stem cells.

The present paper summarizes the results of the second German consensus meeting on immunogenetic donor search for allotransplantation of hematopoietic stem cells held in Essen in November 1999 under the auspices of the German Society for Immunogenetics (DGI) and the German Working Party for Blood and Marrow Transplantation (DAG-KBT). Immunogeneticists and transplant physicians from all over the country agreed to update the national standards for: (1) search strategy including the role of unrelated and extended family donor search after unsuccessful core family donor search, (2) histocompatibility loci to be typed, (3) histocompatibility typing techniques to be used (HLA serology vs DNA-based HLA typing, cellular tests, serum cross-match), and (4) acceptable HLA mismatches in the context of a defined underlying disease, donor type, and conditioning regimen.

Cyclosporine A sensitivity in vitro and P-glycoprotein expression in patients on dialysis and after kidney transplantation.

BACKGROUND: In allogeneic kidney transplantation the response to cyclosporine A (CsA) is important for graft outcome. Although CsA therapy is controlled by drug monitoring to ensure therapeutic CsA levels, the sensitivity to the effects of CsA varies among individuals. Since CsA is an antagonist of cytostatic drugs in P-glycoprotein (Pgp)-mediated transport, increased Pgp expression might contribute to an increased resistance to CsA. METHODS: The sensitivity of lymphocytes at three different concentrations of CsA was tested in a non-radioactive lymphocyte-transformation test and related to Pgp expression as determined by flow cytometry on mononuclear cells. Five groups, including healthy donors (CON; n = 25), patients on dialysis (DIAL; n = 25), patients before transplantation (PTX; n = 5) and after transplantation [short-term (ATX; n = 5) and long-term (LTX; n = 25)] were investigated. RESULTS: In LTX, the sensitivity to CsA at 400 and 1000 ng/ml was significantly different from CON and DIAL. Overall a higher sensitivity to CsA was seen in patients after transplantation. In ATX, sensitivity to CsA was significantly higher than in PTX at a concentration of 1000 ng/ml CsA. However, comparing all groups no significant changes in Pgp expression were noted. Analysing the relationship between CsA sensitivity and Pgp expression, no significant heterogeneity could be observed between the different groups. CONCLUSION: In conclusion, our data suggest that in vitro testing of CsA sensitivity prior transplantation and Pgp expression monitoring yield independent results and cannot substitute for each other as predictors of graft outcome. The differential role of each test for the evaluation of CsA sensitivity or resistance remains to be determined.

Expression of the gene and processed pseudogenes encoding the human and rabbit translationally controlled tumour protein (TCTP).

In humans and rabbits, the TPT1 gene encoding the translationally controlled tumour protein TCTP generates two mRNAs (TCTP mRNA1 and TCTP mRNA2) which differ in the length of their 3' untranslated regions. The distribution of these mRNAs was investigated in 10 rabbit and 50 human tissues. They were transcribed in all tissues investigated, but differed considerably in their quantity and ratio of expression. This indicates an extensive transcriptional control and involvement of tissue-specific factors. In the rabbit genome numerous processed, intronless pseudogenes were detected. Four, corresponding to both types of mRNAs, were sequenced and analysed in detail; all displayed only few mutations and were either preserved completely in the original amino acid sequence of the intron containing gene, or contained only minor mutations in the coding region which did not interrupt the open reading frame. In the mRNA population of rabbit reticulocytes two additional TCTP RNAs of the TCTP mRNA2 type were detected, which have the characteristics of pseudogene transcripts. Pseudogene transcription was supported further by CAT reporter gene assays showing substantial promoter activity of 5'-flanking regions of two TPT1 pseudogenes.

Monocytes from systemic lupus erythematous patients are severely altered in phenotype and lineage flexibility.

OBJECTIVE: Cells of the myeloid lineage comprise a very heterogeneous population with many phenotypes and functional activities including macrophages and dendritic cells. To investigate the status, differentiative potential and lineage commitment of monocytic cells in systemic lupus erythematosus (SLE) patients, this study isolated and cultured peripheral blood monocytes from patients and healthy donors. METHODS: Monocytes were isolated by gradient centrifugation and adherence to plastic dishes. The cells were then cultured for three days, partially supplemented with GM-CSF and interleukin 4 (IL4) to obtain dendritic cells. The differentiation status was monitored by the expression of surface markers using flow cytometry and cytokine secretion. RESULTS: Monocytes from SLE patients expressed significantly lower numbers of the monocytic marker CD14 and HLA-DR while secreting significantly more tumour necrosis factor alpha (TNFalpha) than monocytes from healthy donors. The addition of GM-CSF and IL4 resulted in an inhibition of TNFalpha secretion, but was not sufficient to generate monocytederived dendritic cells. CONCLUSION: Monocytes from SLE patients are severely altered in phenotype and function and have a limited differentiation flexibility towards the accessory type of monocytic cells.

Effects of anti-CD4 antibodies on the release of IL-6 and TNF-alpha in whole blood samples from patients with systemic lupus erythematosus.

Anti-CD4 antibodies have been recently introduced into the therapy of various autoimmune diseases, among them systemic lupus erythematosus (SLE). Their modes of action are not yet fully understood. Interference with cytokine release may be one possible mechanism. Therefore, the effects of anti-CD4 antibodies on the cytokine release of IL-6 (interleukin-6) and TNF-alpha (tumor necrosis factor alpha) were investigated in a whole blood culture system. Basal and phytohemagglutin/lipopolysaccharide (PHA/LPS)-stimulated cytokine patterns were compared to cytokine release after the addition of anti-CD4 antibodies (MAX.16H5) or methylprednisolone in short time whole blood cell culture systems from 12 patients with active SLE, 23 patients with inactive SLE and 12 healthy volunteers. TNF-alpha and IL-6 concentrations were determined in the supernatants by ELISA. High disease activity correlated with an increased production of proinflammatory cytokines. Cell cultures of patients with inactive SLE showed a diminished capacity to respond to mitogenic stimulation. Anti-CD4 antibodies added in vitro suppressed significantly the unstimulated production of IL-6 (P<0.02) in the cell cultures of patients with active SLE and in the PHA/LPS-stimulated cell cultures from both groups of SLE patients (both P<0.001) and healthy volunteers (P<0.01). However, MAX.16H5 did not affect the release of TNF-alpha. In control samples methylprednisolone considerably reduced stimulated and unstimulated IL-6 and TNF-alpha production in all SLE patients, irrespective of the disease state, and in all healthy controls. These data indicate that the proinflammatory cytokines are involved in the pathogenesis of SLE. It is assumed that anti-CD4 antibodies, which can be effective in the treatment of highly active lupus patients, may act via their influence on cytokine release. The decrease of the proinflammatory cytokines IL-6 under therapy with MAX.16H5 could explain the observations of clinical trials and animal studies which showed a reduction of inflammatory parameters and diminished production of autoantibodies following treatment with anti-CD4 antibodies.

The diversity of the lipoxygenase family. Many sequence data but little information on biological significance.

Lipoxygenases form a family of lipid peroxidising enzymes, which oxygenate free and esterified polyenoic fatty acids to the corresponding hydroperoxy derivatives. They are widely distributed in both the plant and animal kingdoms. During the last couple of years more and more lipoxygenase isoforms have been discovered but for most of them the biological significance remains unclear. This review attempts to classify the currently known mammalian lipoxygenase isoforms and critically reviews the concepts for their biological importance.

Complete androgen insensitivity caused by a new frameshift deletion of two base pairs in exon 1 of the human androgen receptor gene.

We describe a novel mutation in exon 1 of the androgen receptor gene in a patient with complete androgen insensitivity (CAIS). Endocrine findings were typical for androgen insensitivity (testosterone serum levels in the upper limit of normal males and increased LH serum concentrations). Biochemical investigations in cultured genital skin fibroblasts of the patient showed a normal 5alpha-reductase activity but a complete absence of androgen binding. Western blot analysis revealed no detectable protein product. Sequence analysis of the entire coding region of the androgen receptor gene resulted in the identification of a 2-bp deletion in codon 472, causing frameshift and introduction of a premature stop codon 27 codons downstream of the mutation.

Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH-GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH-GPx is less well investigated. In this study we cloned the PH-GPx cDNA from a mouse fibroblast cDNA library and the PH-GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5'-flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies.

Tissue-specific translational regulation of alternative rabbit 15-lipoxygenase mRNAs differing in their 3'-untranslated regions.

By screening a rabbit reticulocyte library, an alternative 15-LOX transcript of 3.6 kb (15-LOX mRNA2) was detected containing a 1019 nt longer 3'-untranslated region (UTR2) than the main 2.6 kb mRNA (15-LOX mRNA1). In anaemic animals, northern blotting showed that 15-LOX mRNA2 was predominantly expressed in non-erythroid tissues, whereas 15-LOX mRNA1 was exclusively expressed in red blood cells and bone marrow. The 15-LOX 3'-UTR2 mRNA2 contained a novel 8-fold repetitive CU-rich motif, 23 nt in length (DICE2). This motif is related but not identical to the 10-fold repetitive differentiation control element (DICE1) of 19 nt residing in the 15-LOX UTR1 mRNA1. DICE1 was shown to interact with human hnRNP proteins E1 and K, thereby inhibiting translation. From tissues expressing the long 15-LOX mRNA2, two to three unidentified polypeptides with molecular weights of 53-55 and 90-93 kDa which bound to DICE2 were isolated by RNA affinity chromatography. A 93 kDa protein from lung cytosol, which was selected by DICE2 binding, was able to suppress translational inhibition of 15-LOX mRNA2, but not of 15-LOX mRNA1, by hnRNP E1. A possible interaction between DICE1/DICE2 cis / trans factors in translational control of 15-LOX synthesis is discussed. Furthermore, the 3'-terminal part of the highly related rabbit leukocyte-type 12-LOX gene was analysed. Very similar repetitive CU-rich elements of the type DICE1 (20 repeats) and DICE2 (nine repeats) were found in the part corresponding to the 3'-UTR of the mRNA.

Expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits.

From a rabbit reticulocyte library a full length cDNA was isolated which predicted a novel lipoxygenase (LOX) sharing 99% identical amino acids with the rabbit 15-lipoxygenase. HPLC product analysis of the bacterially expressed protein identified it as a leukocyte-type 12-lipoxygenase (1.12-LOX). This proves the co-expression of a 15-lipoxygenase and a 1.12-lipoxygenase in one mammalian species. Among the six amino acids that are different to rabbit 15-lipoxygenase, leucine 353 is shown to be the primary determinant for 12-positional specificity. In the 3'-untranslated region of the 12-LOX-mRNA a CU-rich, 20-fold repetitive element has been found, closely related to the differentiation control element (DICE) of the rabbit 15-LOX-mRNA which is organized by ten repeats of 19 bases. By genomic PCR the 3'-terminal part of the gene for the novel 12-lipoxygenase containing the introns 10-13 has been amplified and sequenced. The introns were very similar in length to the corresponding 15-lipoxygenase introns with 89% to 95% identical nucleotide sequences. By screening a rabbit reticulocyte library an alternative 15-lipoxygenase transcript of 3.6 kb has been detected containing a 1019 nucleotides longer 3'-untranslated region (UTR2) than the main 2.6 kb mRNA. The determination of the tissue distribution by Northern blotting showed that the 3.6 kb mRNA2 was only expressed in non-erythroid tissues, whereas the 2.6 kb mRNA1 was exclusively expressed in reticulocytes. The only cell type which has been found to express the 1.12-lipoxygenase abundantly are monocytes. The results indicate that the expression of 1.12-lipoxygenase and 15-lipoxygenase is highly regulated. The UTR2 of the 15-LOX-mRNA2 contained a novel eight-fold repetitive CU-rich motif of 23 bases length which is related but not identical to the DICE of 19 bases in the UTR1. The analysis of a genomic recombinant of the complete 9.0 kb Alox15 gene confirmed that UTR1 and UTR2 are not interrupted by an additional intron.

Structure of the promoter and complete sequence of the gene coding for the rabbit translationally controlled tumor protein (TCTP) P23.

We have isolated genomic recombinants containing the complete gene coding for the rabbit translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF) P23. The gene is organized into five introns and six exons and its total length amounts to 3819 nucleotides. All intron/exon boundaries are in accordance with the GT/AG rule. Transcription of the gene generates two mRNAs of 843 and 1163 nucleotides differing in the length of their 3'-untranslated regions. They are formed by alternative polyadenylation. The transcription initiation site has been determined by comparison of sequences of the gene and several processed TCTP pseudogenes. The full-length 5'-untranslated region comprises 116 nucleotides and starts with an oligopyrimidine tract important for translational regulation. Additionally 1.2 kb of the 5'-flanking promoter region has been sequenced. The promoter contains a TATA box at -30 and potential binding sites for transcription factors such as stimulating protein 1 (Sp1), nuclear factor 1 (NF1), activator protein 1 (AP1), c-Ets1, cAMP-response element (CP2), myeloid-specific zinc finger protein 1 (MZF1) and others. For functional analysis 5'-flanking sequences up to -918 were fused to the chloramphenicol acetyltransferase (CAT) gene and tested using a rabbit aortic smooth-muscle cell line by cell transfection and CAT assays. The results confirm that the analyzed gene is the actively transcribed TCTP gene.

Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits.

In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis.