Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite

Baguer-Morvan, France

Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite

Baguer-Morvan, France

Time filter

Source Type

De Tayrac M.,French National Center for Scientific Research | De Tayrac M.,University of Rennes 1 | De Tayrac M.,Rennes University Hospital Center | Roth M.-P.,French Institute of Health and Medical Research | And 24 more authors.
Journal of Hepatology | Year: 2015

Background & Aims Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. Methods We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. Results One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7 × 10-9 and replication p value of 5 × 10-13). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9 × 10-6 and replication p value of 3.2 × 10-6). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p <0.01). Serum iron levels were also associated with fibrosis stage (p <0.0001). Conclusions This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely. © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.


PubMed | Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite, Institute Of Cancerologie Et Dhematologie and Plateforme de Genetique Moleculaire des Cancers INCa
Type: | Journal: BMC cancer | Year: 2016

Despite reported discordance between the mutational status of primary lung cancers and their metastases, metastatic sites are rarely biopsied and targeted therapy is guided by genetic biomarkers detected in the primary tumor. This situation is mostly explained by the apparent stability of EGFR-activating mutations. Given the dramatic increase in the range of candidate drugs and high rates of drug resistance, rebiopsy or liquid biopsy may become widespread. The purpose of this study was to test genetic biomarkers used in clinical practice (EGFR, ALK) and candidate biomarkers identified by the French National Cancer Institute (KRAS, BRAF, PIK3CA, HER2) in patients with metastatic non-small-cell lung cancer for whom two tumor samples were available.A retrospective study identified 88 tumor samples collected synchronously or metachronously, from the same or two different sites, in 44 patients. Mutation analysis used SNaPshot (EGFR, KRAS, BRAF missense mutations), pyrosequencing (EGFR and PIK3CA missense mutations), sizing assays (EGFR and HER2 indels) and IHC and/or FISH (ALK rearrangements).About half the patients (52%) harbored at least one mutation. Five patients had an activating mutation of EGFR in both the primary tumor and the metastasis. The T790M resistance mutation was detected in metastases in 3 patients with acquired resistance to EGFR tyrosine kinase inhibitors. FISH showed discordance in ALK status between a small biopsy sample and the surgical specimen. KRAS mutations were observed in 36% of samples, six patients (14%) having discordant genotypes; all discordances concerned sampling from different sites. Two patients (5%) showed PI3KCA mutations. One metastasis harbored both PI3KCA and KRAS mutations, while the synchronously sampled primary tumor was mutation free. No mutations were detected in BRAF and HER2.This study highlighted noteworthy intra-individual discordance in KRAS mutational status, whereas EGFR status was stable. Intratumoral heterogeneity for ALK rearrangement suggests a limitation of single-biopsy analysis for therapeutic strategy with crizotinib.


Tripathi R.,French Institute of Health and Medical Research | Tripathi R.,University of Western Brittany | Benz N.,French Institute of Health and Medical Research | Culleton B.,Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite | And 4 more authors.
PLoS ONE | Year: 2014

The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future. © 2014 Tripathi et al.


PubMed | French Institute of Health and Medical Research, Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite and University of Western Brittany
Type: Journal Article | Journal: PloS one | Year: 2014

The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.


Bonini J.,French Institute of Health and Medical Research | Bonini J.,Montpellier University | Varilh J.,French Institute of Health and Medical Research | Varilh J.,Montpellier University Hospital Center | And 18 more authors.
Genetics in Medicine | Year: 2015

Purpose:Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations.Methods:We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified.Results:Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF.Conclusion:Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches. © American College of Medical Genetics and Genomics.


Moalic-Allain V.,Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite
Annales de Biologie Clinique | Year: 2014

The LuminexTM technology has become an important tool for HLA antibody screening and identification. This is the most sensitive technology to detect HLA antibodies for transplant patients and patients on awaiting list, and it has ushered a new strategy to determine HLA compatibility between donor and recipient. Moreover, the clinical relevance of all detected anti-HLA antibodies is not well understood, because this technique was shown to be prone to many artefacts or interferences, leading to a complicated interpretation for biologists and clinicians. Our objective in this article is to provide a careful consideration about this solid phase assay, and to focus attention on raised questions about technical performance and interpretation of the results.We should keep in mind that our results could change the clinical management of sensitized patients, their aptitude to receive a graft, and their follow-up.


De Braekeleer M.,University of Health Sciences | De Braekeleer M.,French Institute of Health and Medical Research | De Braekeleer E.,University of Health Sciences | De Braekeleer E.,French Institute of Health and Medical Research | And 10 more authors.
Journal of Biomedicine and Biotechnology | Year: 2011

The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions. © 2011 Etienne De Braekeleer et al.


Moalic V.,Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite
Pathologie Biologie | Year: 2013

Allogeneic haematopoietic stem cell transplantation is the choice treatment for many haematological malignancies. Granulocyte-colony-stimulating factor (G-CSF) has been widely used to mobilize stem cells into the peripheral blood from healthy siblings or volunteer unrelated donors. To a large extent, the use of mobilized peripheral blood haematopoietic stem cells has replaced marrow-derived stem cells as the preferred source of donor haematopoietic stem cells. Clinicians have been aware since the first clinical use, that administration of G-CSF, even in a single short course, could possibly be a risk for healthy donors either in short-term or as a delayed effect. The immediate side effects of G-CSF have been established for a long time, most of them are frequent but transient, self-limited and without long-term consequences. Questions have been raised about potential long-term adverse effects such as an elevated risk of haematological malignancies after G-CSF administration. More long-term safety data from registries are needed to adequately evaluate such a relationship. Our objective in this article is to provide an in-depth review of reported adverse events associated with the use of G-CSF in healthy donors and to focus attention on unanswered questions related to their long-term follow-up. © 2012 Elsevier Masson SAS.


De Braekeleer E.,University of Health Sciences | De Braekeleer E.,French Institute of Health and Medical Research | Meyer C.,Goethe University Frankfurt | Douet-Guilbert N.,University of Health Sciences | And 13 more authors.
Molecular Oncology | Year: 2011

Chromosomal rearrangements involving the MLL gene have been associated with many different types of hematological malignancies. Fluorescent in situ hybridization with a panel of probes coupled with long distance inverse-PCR was used to identify chromosomal rearrangements involving the MLL gene. Between 1995 and 2010, 27 patients with an acute leukemia were found to have a fusion gene involving MLL. All seven ALL patients with B cell acute lymphoblastic leukemia were characterized by the MLL/AFF1 fusion gene resulting from a translocation (5 patients) or an insertion (2 patients). In the 19 AML patients with acute myeloblastic leukemia, 31.6% of all characterized MLL fusion genes were MLL/MLLT3, 21.1% MLL/ELL, 10.5% MLL/MLLT6 and 10.5% MLL/EPS15. Two patients had rare or undescribed fusion genes, MLL/KIAA0284 and MLL/FLNA. Seven patients (26%) had a complex chromosomal rearrangement (three-way translocations, insertions, deletions) involving the MLL gene. Splicing fusion genes were found in three patients, leading to a MLL/EPS15 fusion in two and a MLL/ELL fusion in a third patient. This study showed that fusion involving the MLL gene can be generated through various chromosomal rearrangements such as translocations, insertions and deletions, some being complex or cryptic. A systematic approach should be used in all cases of acute leukemia starting with FISH analyses using a commercially available MLL split signal probe. Then, the analysis has to be completed, if necessary, by further molecular cytogenetic and genomic PCR methods. © 2011 Federation of European Biochemical Societies.


PubMed | Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite
Type: Journal Article | Journal: Annales de biologie clinique | Year: 2016

Current high resolution HLA typing technologies produce ambiguous results, and it is often necessary to perform additionnal tests to resolve these ambiguities. Next generation sequencing is a promising technology, which can overcome this problem. It is going to usher a new strategy to determine HLA compatibility between donor and recipient. It can lead to non ambiguous results by analysing the full amplified sequence of HLA genes and by eliminating heterozygote phase ambiguities. Instead, as many new techniques, we can face several problems, such as analysis difficulties because of incomplete HLA sequences in the database or errors related to the sequencing instrumentation. Moreover, the clinical relevance of analysing non coding regions of HLA genes is not well understood, but raise questions about the interest of getting HLA full sequence to understand drugs side effects or pathogenesis of infectious or auto-immune diseases. Our objective in this article is to present a commercial workflow for HLA typing by NGS, on Ion Torrent PGM sequencer, and to focus attention about pitfalls encountered during the analysis.

Loading Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite collaborators
Loading Laboratoire Of Genetique Moleculaire Et Dhistocompatibilite collaborators