André Le Treut

Integrative genome‐wide analysis reveals a robust genomic glioblastoma signature associated with copy number driving changes in gene expression

Glioblastoma multiforme shows multiple chromosomal aberrations, the impact of which on gene expression remains unclear. To investigate this relationship and to identify putative initiating genomic events, we integrated a paired copy number and gene expression survey in glioblastoma using whole human genome arrays. Loci of recurrent copy number alterations were combined with gene expression profiles obtained on the same tumor samples. We identified a set of 406 “cis‐acting DNA targeted genes” corresponding to genomic aberrations with direct copy‐number‐driving changes in gene expression, defined as genes with either significantly concordant or correlated changes in DNA copy number and expression. Functional annotation revealed that these genes participate in key processes of cancer cell biology, providing insights into the genetic mechanisms driving glioblastoma. The robustness of the gene selection was validated on an external microarray data set including 81 glioblastomas and 23 non‐neoplastic brain samples. The integration of array CGH and gene expression data highlights a robust cis‐acting DNA targeted genes signature that may be critical for glioblastoma progression, with two tumor suppressor genes PCDH9 and STARD13 that could be involved in tumor invasiveness and resistance to etoposide.

Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1 -positive leukemia

BackgroundThe t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR.ResultsWe compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI – highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings.ConclusionGene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE).

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.

Analytical performance of the serum free light chain assay

Abstract Background: The Freelite™ system for nephelometric or turbidimetric measurement of serum free light chains (FLCs) has been available since 2001. It has been valuable for the management of patients with oligosecretory myeloma, light chain myeloma and AL amyloidosis. However, there are several limitations of the method. The goal of this study was to evaluate the analytical performance of the FLC assay. Methods: Titrated controls and clinical serum specimens were used to determine precision and post-dilution recovery. Results: As reported elsewhere, we found that the assay had several limitations, including poor post-dilution linearity and overestimation by nephelometry. Conclusions: These data demonstrate that the results of the FLC assay must be interpreted jointly by the clinician and the biologist, taking into account the individual patients clinical and biological characteristics. Clin Chem Lab Med 2010;48:73–9.

Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis

SummaryTwo yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100kb centromeric to the HLA-A locus, or on the telomeric side.

Serum Ceruloplasmin and Ferroxidase Activity Are Not Decreased in Hepatic Failure Related to Alcoholic Cirrhosis: Clinical and Pathophysiological Implications

BACKGROUND A decrease in serum ceruloplasmin (Cp), a protein involved in iron metabolism through its ferroxidase activity, is classically claimed to be observed in severe hepatic failure of non-wilsonian chronic liver disease and therefore to be a confounding factor for the diagnosis of Wilsons disease. Moreover, a simultaneous decrease in ferroxidase activity could be hypothesized as playing a role in the development of the hepatic siderosis frequently observed in advanced chronic liver diseases. The aim of this study was to test the validity of these two statements. METHODS This study investigated Cp, determined by immunonephelometry, and its ferroxidase 1 activity determined by Erels method in 33 male patients with severe alcoholic cirrhosis compared with 66 healthy male volunteers, selected on strict criteria. Each patient was age-matched with two controls. Nonparametric tests were used for statistical analysis. RESULTS The mean values of Cp were significantly higher in cirrhotic patients as compared with control subjects. A significant elevation of Cp was also observed in the subgroup of 11 cirrhotic patients who had normal serum C-reactive protein levels. The mean values of ferroxidase 1 activity were similar to those obtained in control subjects. CONCLUSIONS Low serum Cp should not be expected in severe hepatic cirrhosis of non-wilsonian origin. Hepatic siderosis in advanced chronic liver disease is likely to be unrelated to decreased ferroxidase activity.

Evaluation of ETF1/eRF1, mapping to 5q31, as a candidate myeloid tumor suppressor gene

Interstitial deletion of the long arm of chromosome 5 is a recurrent abnormality, mainly associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and it has been proposed therefore that the deleted region may contain a myeloid tumor suppressor gene. We have recently mapped a human translation termination factor gene, ETF1, to band 5q31 at D5S500, and thus to the smallest commonly deleted segment. We have evaluated ETF1 as a candidate myeloid tumor suppressor gene by analysis of the human acute myeloid leukemia cell line HL60, and of patients suffering from malignant myeloid diseases with cytogenetically-defined abnormalities of chromosome 5. Fluorescence in situ hybridization analysis revealed hemizygous loss of the ETF1 locus in HL60 cells and in four of five leukemic samples, but no inactivating mutations were identified by sequencing of the remaining ETF1 allele.

Serum ceruloplasmin and ferroxidase activity are decreased in HFE C282Y homozygote male iron-overloaded patients

BACKGROUND/AIMS A body of evidence suggests that ceruloplasmin (Cp), the major serum copper-containing protein, acts in iron metabolism due to its ferroxidase activity which appears essential for iron movements and exchanges. METHODS The present study investigated the serum levels of Cp and its ferroxidase activity in 53 C282Y homozygote genetic hemochromatosis (38 iron overloaded, 15 iron depleted) patients as compared to age and sex-matched healthy volunteers. RESULTS Serum levels of Cp were significantly decreased in iron-overloaded male hemochromatotic patients vs. the control group (P=0.02). Furthermore, serum ferroxidase activity was strongly and significantly lower in iron-overloaded male hemochromatotic patients (P<0.001). In contrast, in iron-depleted male hemochromatotic patients, who were under maintenance therapy by regular phlebotomies, serum levels of Cp and ferroxidase activity were not statistically different from those observed in controls. CONCLUSIONS These data: (i) show that serum Cp and ferroxidase activity are decreased when C282Y homozygote men are iron overloaded and normal when iron depleted; (ii) suggest that iron may modulate the Cp gene expression; and (iii) raise the issue of the putative role of decreased serum ferroxidase activity in the phenotypic expression of HFE-1 hereditary hemochromatosis.

Don't forget to test for D-lactic acid in short bowel syndrome

inflammation parameters, a reduction in prednisolone dose below 25 mg/day was not possible. Simultaneous treatment with azathioprine with doses of up to 200 mg/day was started and the cyclophosphamide interval was lengthened. The patient experienced opportunistic infections such as herpes zosteroticus and mucosal candidiasis. Four months later, in September 1998, a relapse with orthopnea, bizarre interstitial lung infiltrations, recurrent vomiting, and headaches occurred. Gastroscopy revealed chronic active H. pylori negative gastritis while cranial nuclear magnetic resonance imaging showed no vasculitis-like lesions. Additionally, he now had persistent hematuria with dysmorphic red cells and acanthocytes, a reduced creatinine clearance, proteinuria, indicating glomerulonephritis. Renal biopsy was refused and antinuclear and anticytoplasmic antibodies were negative. At this point, MMF 1 g was givenb.i.d. and well tolerated by the patient. Over the next 9 months inflammation parameters and liver enzymes normalized, a CT-scan of the lung showed minimal fibrosis without any further infiltration, and spirometry/arterial O 2-tension improved. With the exception of the osteoporosis and the persistent neurological deficit, the patient remained asymptomatic during the reduction of prednisolone to 7.5 mg/day. The diabetes mellitus and arterial hypertension could once again be controlled. Creatinine clearance improved while signs of chronic glomerulonephritis persisted. Follow-up after 1 yr revealed stable lung disease and left-sided clinically inactive colitis. Several factors might lead to airway manifestations (1): Concurrent smoking was believed to be a cofactor but fewer than expected current smokers were found among UC patients. Despite scarce data, positive antinuclear or anticytoplasmic antibodies were found equally often in UC patients with and without lung manifestation. Surprisingly, no correlation between the intestinal activity of UC and frequency or severity of the lung disease was observed. This finding is supported by the fact that colectomy has no influence on lung manifestation (1). Drugs such as sulfasalazine and 5-ASA can also induce eosinophilic lung infiltrations, but the proportion of patients on these drugs in both groups (with and without lung manifestation) was identical (1). Our patient took 5-ASA only until 1996 and showed no eosinophilic changes. Generally, there are no treatment recommendations for UC patients with a lung manifestation unresponsive to steroids or for those needing prolonged treatment. Adding azathioprine as a steroid-sparing immunosuppressive appears logical but even in high doses it does not always act quickly, as this case exemplifies. We instituted cyclophosphamide (as in patients with Wegener’s granulomatosis) due to the impression that this syndrome could just as well be related to systemic vasculitis with bowel manifestation (highlighted by renal involvement) as to UC. Despite initial success with cyclophosphamide, the achievement of a stable remission remained a problem. Cyclosporine, as another alternative, is not supported by enough evidence to warrant its use to maintain remission (2). Being well established as an immunosuppressive agent after transplantation and despite two small promising studies in patients with Crohn’s disease (3–5), MMF is an experimental drug in the treatment of inflammatory bowel disease. It may be a valuable treatment alternative for patients with severe UC and/or UC with pulmonary involvement but should be reserved for selected cases unresponsive to other therapies (3).

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.