A polymorphic Alu insertion that mediates distinct disease-associated deletions.

Large deletions that are associated with insertions of Alu-derived sequence represent a rare, but potentially unique class of alterations. Whether they form by a one-step mechanism or by a primary insertion step followed by an independent secondary deletion step is not clear. We resolved two disease-associated SPAST deletions, which involve distinct exons by long range PCR. Alu-derived sequence was observed between the breakpoints in both cases. The intronic regions that represent the targets of potentially involved Alu retrotransposition events overlapped. Microsatellite- and SNP-based haplotyping indicated that both deletions originated on one and the same founder allele. Our data suggest that the deletions are best explained by two-step insertion-deletion scenarios for which a single Alu retrotransposition event represents the shared primary step. This Alu then mediated one of the deletions by non-homologous end joining and the other by non-allelic homologous recombination. Our findings thus strongly argue for temporal separation of insertion and deletion in Alu insertion-associated deletions. They also suggest that certain Alu integrations confer a general increase in local genomic instability, and that this explains why they are usually not detected during the probably short time that precedes the rearrangements they mediate.

Functional mutation analysis provides evidence for a role of REEP1 in lipid droplet biology.

Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

Mass spectometry-based protein patterns in the diagnosis of sepsis/systemic inflammatory response syndrome.

Early differential diagnosis of systemic inflammatory reactions in critically ill patients is essential for timely implementation of lifesaving therapies. Despite many efforts made, reliable biomarkers to discriminate between infectious and noninfectious causes of systemic inflammatory response syndrome (SIRS) are currently not available. Recent advances in mass spectrometry-based methods have raised hopes that identification of spectral patterns from serum/plasma samples can be instrumental in this context. We compared protein expression patterns from patients with SIRS of infectious and noninfectious origin. Plasma samples from 166 patients obtained under rigorously standardized preanalytical conditions were applied to Q10 and CM10 ProteinChips. Protein profiles were used to train and develop decision tree classification algorithms. Discriminatory peaks were isolated and identified. Classification trees distinguished patients with noninfectious SIRS with organ dysfunction following open heart surgery using cardiopulmonary bypass from those with severe sepsis or septic shock with distinct sensitivities and specificities. Results were validated in a blinded test set in two independent experiments and in a second independently collected test set. Discriminatory peaks at 13.8 and 55.7 kd were identified as transthyretin and α1-antitrypsin; the third protein at m/z 4,798 was assigned to a proteolytic fragment of α1-antitrypsin. Taken together, our data demonstrate that plasma protein profiling allows reproducible discrimination between patients with infectious and noninfectious SIRS with high sensitivity and specificity. However, rigorous standardization as well as considering drug-related interferences is essential when interpreting protein profiling studies. Identification of discriminatory proteins suggests a direct link between infectious-related protease activity and a sepsis-specific diagnostic pattern for discrimination of patients with SIRS.

Immediate diagnosis of ventriculits: evaluation of parameters independent of microbiological culture.

BACKGROUND: Generally accepted reference values in CSF diagnostics are not valid in cerebrospinal fluid (CSF) containing large amounts of blood. Residual blood may obscure ventriculitis as diagnostics largely depend on parameters such as cell count, lactic acid and total protein measurement. We sought to improve the diagnostics by evaluating a cytokine panel and soluble CD62L as markers of ventriculitis. In addition, we tested an algorithm of established parameters to predict ventriculitis in a specific patient collective. METHODS: Analysis was performed on ventricular CSF samples from 50 consecutive patients. Gram staining, microbiological culture, total cell count, total protein and CD62L expression on neutrophil granulocytes were analysed immediately. Cytokines and soluble CD62L were measured by flow cytometry. FINDINGS: Positive culture was detected in ten patients. Of all parameters tested only IL1-beta, IL8 and CD62L on neutrophils were significantly different between culture-positive and -negative patients. The highest predictive value was obtained when analysing IL1-beta. The predictive value of a parameter combination (IL6 in CSF, C-reactive protein and leukocytes in periphereal blood) was comparable to IL1-beta. Half of the patients in this analysis were identified as culture positive because of the lack of inflammatory response. CONCLUSIONS: IL1-beta and perhaps also IL8 provide very good analytical performance when looking for ventriculitis in patients with residual blood in CSF. Turn-around time is short, and results could be reported within 1 h for 24 h a day. In some patients application of glucocorticoids may result in restricted inflammatory response. Even in these patients IL1-beta provides a reliable parameter for the immediate diagnosis of ventriculitis.

Methylation-specific multiplex ligation-dependent probe amplification in meningiomas.

Genomic loss and promotor methylation contribute to inactivation of tumor suppressor genes (TSGs). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a relatively new method for simultaneous detection of both these alterations. Here, we apply MS-MLPA to a series of 15 meningiomas of different WHO grades. The two MS-MLPA probe sets used detect copy number changes in 55 unselected TSGs and promotor methylation in a subset of 36 of these genes. Our findings concerning genomic deletions are concordant with previously published studies using alternative techniques. The number of aberrations identified per tumor increased with histopathologically determined grading. The most frequent single event was deletion of the von Hippel-Lindau (VHL) gene in 12 of the 15 tumors. Moreover, VHL deletion status was associated with presence/absence of peritumoral edema. Methylation was rare, being observed in only four tumors and in each case restricted to a single gene. We conclude that a meningioma-specific MS-MLPA probe set would be a valuable tool for both research and diagnostic approaches in these tumors.

Ciprofibrate, clofibric acid and respective glycinate derivatives. Effects of a four-week treatment on male lean and obese Zucker rats.

Fibrates are widely prescribed in hyperlpidemic patients to prevent atherosclerosis. Their therapeutic use, however, can be associated with adverse effects like gastrointestinal disorders, myalgia, myositis and hepatotoxicity. In rodents large doses can even cause hepatocellular carcinoma. Additionally, interactions with the biotransformation of other compounds at the cytochrome P450 (CYP) system have been observed. Thus, the discovery of new substances or derivatives with less side effects is of great interest. In the present study the influence of a four-week daily oral administration of 2 mg/kg body weight ciprofibrate (CAS 52214-84-3) or of 100 mg/kg body weight clofibric acid (CAS 882-09-7) was compared to that of the respective doses of their newly synthesized glycine conjugates in adult male lean and obese Zucker rats. Although obese rats displayed distinctly higher serum lipid concentrations, after fibrate treatment values were significantly lowered in lean animals only. Livers of obese rats were significantly enlarged, histologically showing a fine-droplet like fatty degeneration and an increase in glycogen content, but no signs of inflammation. After fibrate administration histologically a hypertrophy, an eosinophilia, a reduced glycogen content and also hepatocyteapoptosis were observed. Livers of obese rats displayed higher CYP1A1 andCYP2E1 expression, but lower immunostaining for CYP2B1 and CYP3A2. No differences between the two groups of rats were seen with respect to CYP4A1 expression. Due to fibrate treatment especially CYP2E1 and CYP4A1, but also CYP1A1, 2B1 and 3A2 were induced. Resulting CYP mediated monooxygenase activities were also elevated in most cases. In general, effects of clofibric acid and clofibric acid glycinate (CAS 4896-55-3) were less distinct than those of ciprofibrate and its glycinate (CAS 640772-36-7). With no parameterinvestigated major differences were seen between the parent fibrates and their glycine conjugates. Thus, the present investigations revealed no noticeable advantages of the glycinates over ciprofibrate or clofibric acid.

A multi-exonic SPG4 duplication underlies sex-dependent penetrance of hereditary spastic paraplegia in a large Brazilian pedigree.

SPG4 mutations are the most frequent cause of autosomal-dominant hereditary spastic paraplegia (HSP). SPG4 HSP is characterized by large inter- and intrafamilial variability in age at onset (AAO) and disease severity. The broad spectrum of SPG4 mutations has recently been further extended by the finding of large genomic deletions in SPG4-linked pedigrees negative for 'small' mutations. We had previously reported a very large pedigree, linked to the SPG4 locus with many affected members, which showed gender difference in clinical manifestation. Screening for copy number aberrations revealed the first case of a multi-exonic duplication (exon10_12dup) in the SPG4 gene. The mutation leads to a premature stop codon, suggesting that the protein product is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. The present family suggests that this HSP pathogenesis may be modulated by factors related to individual background and gender as observed for other autosomal dominant conditions, such as facio-scapulohumeral muscular dystrophy or amyloidosis. Understanding why some individuals, particularly women, are 'partially protected' from the effects of this and other pathogenic mutations is of utmost importance.

Automated monitoring of C2 and C0 blood levels of mycophenolic acid and cyclosporine on the Abbott Architect c8000.

OBJECTIVES: Evaluation of the performance of the EMIT 2000 Cyclosporin assay using 2 sets of assay calibrators and the EMIT 2000 mycophenolic acid assay to measure C0 and C2 concentrations on the Abbott Architect c8000 analyzer. DESIGN AND METHODS: Imprecision studies were performed. Cyclosporin concentration was assayed by EMIT on the c8000, by ACMIA on the Dimension and by LC-MS/MS while mycophenolic acid was analyzed by EMIT on c8000 and on Dimension and by HPLC. RESULTS: Agreement between cyclosporin and mycophenolic acid concentrations assayed on the c8000 and on the Dimension was very good. Method comparison between the c8000 and LC-MS/MS resulted in a relative bias of 15.7% for C0 and 11.5% for C2 concentrations. Relative bias of the mycophenolic acid concentrations assayed on the c8000 and the HPLC was 37.7%. CONCLUSIONS: When reported properly to the clinician mycophenolic acid and cyclosporine blood levels can be monitored using the EMIT assays on the c8000 consolidating standard routine workflow and reducing reagent costs significantly.

A paediatric supratentorial primitive neuroectodermal tumour associated with malignant astrocytic transformation and a clonal origin of both components.

The case of a 7-year-old boy suffering from a supratentorial primitive neuroectodermal tumour (sPNET) at the age of 5 is presented. The tumour has been characterized by astrocytic areas within the sPNET revealing malignant transformation up to a multiform glioblastoma during the course of the disease. The clonal origin of both tumour components was established by loss of heterozygosity (LOH) analysis. Clinically, the tumour showed an aggressive biological behaviour with two recurrences. We discuss this very rare case and the first description of the clonal origin of distinct and distinguishable tumour components taking into consideration published literature.

Outcome-based profiling of astrocytic tumours identifies prognostic gene expression signatures which link molecular and morphology-based pathology.

Astrocytomas are intracranial malignancies for which invasive growth and high motility of tumour cells preclude total resection; the tumours usually recur in a more aggressive and, eventually, lethal form. Clinical outcome is highly variable and the accuracy of morphology-based prognostic statements is limited. In order to identify novel molecular markers for prognosis we obtained expression profiles of: i) tumours associated with particularly long recurrence-free intervals, ii) tumours which led to rapid patient death, and iii) tumour-free control brain. Unsupervised data analysis completely separated the three sample entities indicating a strong impact of the selection criteria on general gene expression. Consequently, significant numbers of specifically expressed genes could be identified for each entity. An extended set of tumours was then investigated by RT-PCR targeting 12 selected genes. Data from these experiments were summarised into a sample-specific index which assigns tumours to high- and low-risk groups as successfully as does morphology-based grading. Moreover, this index directly correlates with definite survival suggesting that integrated gene expression data allow individualised prognostic statements. We also analysed localisation of selected marker transcripts by in situ hybridization. Our finding of cell-specificity for some of these outcome-determining genes relates global expression data to the presence of morphological correlates of tumour behaviour and, thus, provides a link between morphology-based and molecular pathology. Our identification of expression signatures that are associated individually with clinical outcome confirms the prognostic relevance of gene expression data and, thus, represents a step towards eventually implementing molecular diagnosis into clinical practice in neuro-oncology.

Single and chronic administration of ciprofibrate or of ciprofibrate-glycinate in male Fischer 344 rats: comparison of the effects on morphological and biochemical parameters in liver and blood.

Fibrates lead to a reduction of serum triglycerides and cholesterol in hyperlipidemic patients. Their therapeutic use, however, can be associated with adverse effects like gastrointestinal disorders, myalgia, myositis and hepatotoxicity. Large doses can even cause hepatocellular carcinoma in rodents. Additionally, interactions with the biotransformation of other compounds at the cytochrome P450 (CYP) system have been observed. Thus, the discovery of new derivatives with less of these side effects is of great interest. In the present study a single (10 mg/kg body weight) or a 4-week (1 or 10 mg/kg body weight daily) oral administration of ciprofibrate or of the newly synthesized ciprofibrate-glycinate was investigated in adult male Fischer 344 rats. Serum lipid concentrations were distinctly decreased after single but only slightly after chronic administration of the two fibrates, whereas liver parameters revealed a slight concentration and time dependent hepatotoxicity. Histologically, a hypertrophy, an eosinophilia, a reduced glycogen content and also an apoptosis of the hepatocytes was observed. Effects were more pronounced after chronic treatment and after application of the higher dosage. All CYP enzymes investigated were induced in a time and concentration dependent manner. Resulting CYP mediated monooxygenase and oxidase activities showed a dependency both on enzyme induction and hepatotoxic effects. With no parameter investigated major differences were seen between ciprofibrate and ciprofibrate-glycinate. Thus, the present investigations revealed no noticeable advantages of ciprofibrate-glycinate over its parent compound ciprofibrate.

Low expression but infrequent genomic loss of the putative tumour suppressor DBCCR1 in astrocytoma.

DBCCR1 (deleted in bladder cancer chromosomal region 1) has been reported as the gene functionally affected by frequent loss of 9q32-33 in transitional cell carcinomas of the urinary bladder. For these particular tumours, its proposed role in tumour suppression is supported both by the observation of methylation-based silencing of DBCCR1 in a large fraction of bladder cancers and by re-expression studies in bladder cancer-derived cell lines. A more general involvement of DBCCR1 in tumour development might be inferred from recent chip-based expression studies in other tumours. The present study addressed expression of DBCCR1 in gliomas, specifically in astrocytomas, using semi-quantitative RT-PCR on 25 tumours of different malignancy grade and on 5 control brain tissue samples. Genomic deletion of the DBCCR1 locus at 9q32-33 was also investigated, together with the CDKN2A locus at 9p21, by loss of heterozygosity analysis in a second series of 26 astrocytic tumours. We found that DBCCR1 mRNA expression is markedly reduced in the majority of tumour samples compared to controls, and that this reduction significantly correlates with tumour grade. Genomic loss of the DBCCR1 region was found in only 5 of 24 (21%) informative samples, with no obvious correlation to tumour grade, while loss of the CDKN2A locus was observed in 13 of 21 (62%) informative samples with high-grade tumours being affected more often. If present, LOH at 9q coincided with LOH at 9p and is then likely to reflect loss of the entire chromosome rather than a specific, potentially causative event. In contrast to the situation in bladder cancer, the prevalent inactivation of DBCCR1 seen at the expression level in astrocytomas is not primarily caused by genomic loss of the gene. Our findings support a more general role for DBCCR1 in tumour suppression with mechanisms of inactivation differing between tumour types.

Rapid generation of detailed loss of heterozygosity profiles for routine diagnosis of gliomas.

Aberrations in the genomes of gliomas seem to correlate well with clinical parameters. Pertinent studies, however, rely on highly sophisticated methods, they require large amounts of high-quality sample material and/or they demand profound analytical expertise. Consequently, molecular tumour analysis has not yet been widely implemented in routine laboratory applications. We have developed an easy-to-perform approach for the rapid derivation of a detailed loss-of-heterozygosity profile from individual gliomas. DNA of PCR quality is extracted in a one-step procedure from routinely obtained material. A microsatellite-based marker set is used to detect the deletion status of genomic regions (i) with established diagnostic relevance, (ii) recurrently found deleted in gliomas, or (iii) generally associated with tumour suppressor activity. The complete profile comprises 25 regions and is generated from 64 markers multiplexed into 18 reactions. Illustratively, we present findings from an anaplastic oligodendroglioma; the molecular data for this tumour allow refined diagnostic and prognostic statements that could not be derived from histology alone. Our approach should prove useful in the routine diagnosis of gliomas. Simultaneously, research data for many highly relevant regions are generated in a comparatively simple and inexpensive way.

Identification of nuclear localisation sequences in spastin (SPG4) using a novel Tetra-GFP reporter system.

Mutations in the human spastin gene (SPG4) cause the most prevalent form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterised by progressive weakness and spasticity of the lower limbs. We address the question of intracellular localisation of spastin. Using polyclonal antibodies against N-terminal spastin sequences, we find that the native protein is localised in both the perinuclear cytoplasm and the nucleus. To identify structural motifs within the protein that can explain entry into the nucleus, we developed a reporter system to test nuclear localisation sequence (NLS)-functionality based on four in-frame fused copies of green fluorescent protein. Using this novel tool we demonstrate that spastin carries two NLSs located in exons 1 and 6. Both are independently functional in mediating nuclear entry.

Analysis of the CTNS gene in patients of German and Swiss origin with nephropathic cystinosis.

The autosomal recessive lysosomal storage disorder, nephropathic cystinosis is characterized by impaired transport of free cystine out of lysosomes. The gene responsible for cystinosis, CTNS, consists of 12 exons and encodes a 55 kDa putative lysosomal membrane protein, called cystinosin. Up to now more than 55 different CTNS mutations have been described in cystinosis. We have analyzed the mutation pattern in a population of 40 cystinosis patients from 35 families of German and Swiss origin. CTNS mutations in 68 out of 70 alleles were identified. The common 57-kb deletion accounted for 65% of the alleles. In five patients we found a known GACT deletion at position 18-21. In two patients we identified a nucleotide substitution at codon 339 and one patient showed a CG insertion at position 697-698. In five patients we observed a G insertion at position 926-927. Moreover, five novel mutations including two deletions involving exon 3 (61-61+2delGGT) and exon 6 (280delG), two insertions in exon 6 (292-293insA) and exon 7 (684insCACTT) and one nucleotide substitution in exon 11 (923G>T) have been identified. These data provide a basis for routine molecular diagnosis of cystinosis in the central European population, especially in cystinosis patients of German and Swiss origin.