News & updates

M12 Full version

Project Update November 2010 - Fullversion

Introduction: The BIO-NMD project is a collaboration of 12 European partners working together with the common aim of trying to identify possible biomarkers in people with neuromuscular disease. Once the potential biomarkers are identified, they will be validated in animal and cell models and in human body fluids. The biomarkers will then be validated according to EMEA rules and regulations.

The project is currently in its 12th month and this report summarises the activities of the partners since the last report in M6. It is intended only as a brief update and summary.


University of Ferrara (UNIFE)
Leader: Alessandra Ferlini

Summary: The University of Ferrara is coordinating the BIO-NMD project. In addition, they are trying to discover biomarkers by looking at DNA and RNA. The team is also involved in providing patients' samples.

Update:  During the last six months UNIFE has:
• started to collect samples and clinical data regarding the categories of patients that should undergo genomic biomarkers discovery – for example, symptomatic and asymptomatic female carriers, DMD patients who are responsive and non-responsive to corticosteroids.
• finalized the design strategy for sequence capturing systems of the gene lists found to be associated with DMD and COL6 myopathies by ARIADNE software. This is together with Applied Biosystems (AB).
• completed the analysis of whole genome transcriptome microarrays performed on WT and Col6a1 -/- mice treated with Ciclosporine A provided by WP 5 Leader, Paolo Bonaldo (UNIPD). These data will be presented in Montpellier in December 2010.
• set-up the first RNA-seq experiment by analyzing RNA from the muscles of three patients with different COL6 related phenotypes treated with Ciclosporin A. Data analysis is in progress.


Leiden University Medical Centre (LUMC)
Leader: Peter Bram ‘t Hoen

Summary: LUMC is looking for biomarkers in cells, muscle tissue and body fluids.

Update: In a search for candidate DNA biomarkers for Duchenne Muscular Dystrophy, LUMC discovered a DNA variant (SNP for single nucleotide polymorphism) associated with the age at which Duchenne patients lose ambulation. This variant could point at a biochemical mechanism that modifies disease progression. This finding was done in a limited set of 70 DNA samples collected and analyzed at LUMC and needs confirmation in larger and independent patient cohorts. To this end, other BIO-NMD partners (Newcastle, London, Ferrara and Montpellier) sent in DNA samples from their Duchenne cohorts for genotyping. The genotyping of these samples has been completed and data analysis is ongoing. This example shows the obvious need for and success of collaboration between the different partners participating in BIO-NMD.

LUMC screened the serum of Duchenne patients for candidate protein biomarkers with ELISA – an immunosorbent assay that is based on antibody detection. They found two proteins with elevated levels in DMD patients compared to healthy age-matched controls and are busy replicating these findings in new cohorts, collected by different BIO-NMD partners.

LUMC used Bruker’s ClinProt system to screen serum of DMD patients and healthy controls for potential new protein and peptide biomarkers. The technology is based on the capture of specific protein subfractions of the serum with functionalized magnetic beads. Weak cation exchange and C18 beads were used to capture hydrophilic and hydrophobic proteins, respectively. They analyzed these subfractions by mass spectrometry and evaluated different analysis strategies, the results of which were laid down in a manuscript. This research so far identified one clear candidate biomarker, represented by different peaks in both the weak cation exchange and C18 subfractions. Validation of this potential biomarker is ongoing.

Since the ClinProt system is highly biased towards high abundant proteins, LUMC evaluated different methods for depletion of these high abundant proteins, and found Biorad’s Proteominer kit to provide most promising results. To obtain a more comprehensive view on the content of medium to low abundant proteins in the serum, they applied further fractionation steps, trypsin digestion and tandem mass spectrometry technology. So far, they have optimized methodologies using serum from healthy subjects and are now ready to analyze sera from DMD and COL6 patients.


University of Newcastle upon Tyne (UNEW)
Leader: Volker Straub

Summary: The Institute for Human Genetics at Newcastle University is involved in three areas of the project: organising the collection and use of patients’ samples; social and ethical aspects of the project; and dissemination of information.

Update: WP1: The provision of a large number of human biomaterials is an essential prerequisite for the biomarker discovery programme of the BIO-NMD project.  As leaders of WP1, UNEW has to improve the availability, exchange and use of human biomaterials for biomarker research by the BIO-NMD partners UCL, UNIFE and UNEW.

During the London meeting in July 2010, the BIO-NMD partners reached a consensus on the need to adhere to the proceedings of the clinical working group, detailed in the WP1 M6 presentation available on the project intranet.  Another important point highlighted in the meeting was the urgent need to determine muscle sample availability across partners and assess the feasibility of parallel studies in which DNA, RNA, plasma and serum are used from the same patient.

UNEW has almost completed the muscle biopsy availability audit – a full list of DMD samples is still to come from UCL - whereby a significant number of biomaterials have been identified across the three centres (UNIFE, UCL and UNEW).  At this juncture, UNEW is in the process of arranging transfer of its muscle samples to partners. UNEW will also present the results in the Montpellier meeting in December 2010 and explore how the BIO-NMD partners wish to best utilise and share these samples.  A further important aspect of the audit was looking at the feasibility of parallel studies using serum, plasma, DNA and RNA from the same patients. A few cases have been identified in this audit and UNEW will discuss with partners how best to use these precious samples.

In terms of new samples collection, UNEW has successfully collected and distributed a significant number of DNA, plasma and serum samples to its partners (LUMC and UNIFE) and provided comprehensive clinical data on each patient. UNIFE and UCL have also contributed samples.  UNEW has also shared these clinical data with WP6 (Bioinformatics) as an example of what should be included in the BIO-NMD database. UNIFE, UCL and INSERM have shared Duchenne DNA samples from their local archive.
 
Regular exchange of samples is anticipated over the coming months.  Consistent communication among partners will be essential in achieving this.  Clearly, it is plausible that samples from certain subgroups identified by the clinical working group will be difficult to obtain.  In this instance, reaching out to EuroBioBank and patients’ registries through the TREAT-NMD platform would be an alternative route.
 
WP8 and 9: In M9, a newsletter was been produced, aimed at patients and which focussed on the ethical use of biomaterials in biobanks. This was distributed through TREAT-NMD and BIO-NMD’s networks as well as by MDC, ENMC, UPPMD, Cure-CMD and it was featured in Orphanews Europe, 20 October 2010. It is hoped that this will remain relevant for several months and can be used as an introduction to BIO-NMD in lay-terms. The second issue will be produced in M18.

The website continues to be promoted and updated with current visits numbering more than 2600 since April 2010. The patient section of the website – a potentially valuable tool for reaching a lay audience – has now been translated into Italian, French, German and Dutch, and more languages will be sought.

The Project Ethics Board (PEB) has met via teleconference and the minutes will soon be circulated according to the agreement made in London that they should be public unless there are specific confidentiality issues. The PEB did not identify or receive any ethical questions generated by BIO-NMD at this meeting.


University of Padova (UNIPD)
Leader: Paolo Bonaldo

Summary: The University of Padova is working on biomarkers in animal models.

Update: During the last 6 months (M6-M12) UNIPD has:

• Investigated the role of oxidative stress and abnormal production of reactive oxygen species (ROS) in the pathophysiology of different muscular dystrophies (MDs), using mdx mice (as model for Duchenne MD) and Col6a1 knockout mice (as model for COL6 diseases) and in collaboration with Fabio Di Lisa’s lab (UNIPD). Pargyline, a MAO inhibitor, was used to reduce ROS production by mitochondria and displayed beneficial effects on the dystrophic phenotype of both Col6a1 knockout and mdx mice. Oxidation of myofibrillar proteins, as probed by formation of disulphide cross-bridges, was detected in both Col6a1 knockout and mdx muscles and was significantly reduced by pargyline treatment along with myofiber apoptosis, leading also to increased muscle strength. MAO activity and ROS-dependent oxidation of myofibrillar proteins represent novel biomarkers for multiple forms of MDs, and it will be relevant to assess their usefulness in patients.

• Performed a thorough study of the autophagic machinery in Col6a1 knockout mice and in Bethlem and Ullrich patients. This study revealed that skeletal muscles of Col6a1 knockout mice have impaired autophagic flux, characterized by decreased LC3 lipidation, lower induction of Beclin-1 and Bnip3, and lack of autophagosomes both in physiological conditions and after starvation. Forced reactivation of autophagy by genetic, nutritional and pharmacological approaches is able to restore myofiber survival and ameliorate the dystrophic phenotype. Some of the key proteins involved in autophagy (such as LC3, Beclin-1 and Bnip3) may be good candidates as novel biomarkers for the early detection of muscle defects in COL6 diseases.

• Started to investigate the regenerative potential of muscles of Col6a1 knockout mice in basal conditions and in response to muscle injury, with the aim of identifying additional pathophysiological biomarkers to be translated into humans.


Institute of Child Health, University College London (UCL)
Leader: Francesco Muntoni

Summary: UCL Institute of Child Health will be involved in the collection and distribution of biomaterials for the research (e.g. muscle cells, blood and urine from Duchenne muscular dystrophy patients). They have access to these samples via the MRC Centre for Neuromuscular Diseases biobank. 

They will also be involved in the search for both genetic and protein biomarkers that would indicate differences in disease progression and response to potential drugs.  The goal is to have a non-invasive way of monitoring disease severity or treatment benefit, e.g. by measuring levels of a protein in the blood or urine.

Update:  During the second six months UCL has:

• Audited the available muscle biopsy samples from DMD and COL IV patients as well as the DNA samples from DMD patients. Approximately 200 muscle biopsies of DMD and 40 muscle biopsies of COL VI related disorders were checked.
• Compiled a list of patients that are going to be included into the study. These patients were stratified according to the study criteria.
• Completed technical preparation in collaboration with UCL genomics for the next generation sequencing sample preparation, whole exome and RNA-seq. Several protocols were examined and the appropriate ones were selected.
• Commenced collection of plasma and serum from DMD patients.
• Attended (Dr. Zaharieva ) the Next Generation Sequencing data analysis course (1-3 November 2010 in Leiden) and the Next Generation Sequencing Congress (15th & 16th November 2010, London Heathrow) where a poster on BIO-NMD genomic biomarkers discovery was presented.


University of Rome Tor Vergata (UNIROMA)
Leader: Giuseppe Novelli

Summary: The university will be responsible for monitoring the results obtained across the labs involved in the BIO-NMD project. They will ensure that the methods used follow rules set out by the European Medicines Agency (EMEA).
 
Update: At the last meeting of the BIO-NMD project (London, 8-9 July 2010) UNIROMA illustrated some guidelines of EMA (European Medicine Agency) about the definition of Genomic Biomarkers (GB) and about procedures regarding the application for qualification processes of  these biomarkers in the diagnostic and drug development fields. They also presented the possibility for the Consortium to apply for “Scientific Advice” or a “Briefing Meeting” at the EMA in the future.
Now UNIROMA is also monitoring and following the drafting of news documents (in progress) by EMA that could be useful for the various WPs’ work, in order to highlight them to the partners if approved.

Institut National de la Santé et de la Recherche Médicale (INSERM)
Leader: Christophe Béroud

Summary: INSERM is the leader of WP 6 - Bioinformatics tools for identifying functional pathways, potential targets and data outflow integration - and will also be involved in WP 2 - Genomic biomarkers discovery by genome-wide analyses of DNA and RNA and WP 4 - Exploratory biomarkers validation in humans.
They will provide the project with patient cohorts and expression analysis in order to validate the specificity of discovered biomarkers (WP2). They will also participate in the in vitro validation of genetic variations (WP4). As part of WP6, INSERM will create new bioinformatics tools and databases to handle data generated during the project.

Update: Since the Biomarkers discovery process involves different fields (Genomic, Transcriptomic and Proteomic), in order to harmonize data collection and integration, a first step was to identify key tools used by partners and experts as well as the data structure generated by these various high throughput approaches. A questionnaire has been sent to all partners and was used to define the database structure as well as the needs for data manipulation (translation in different formats) prior to their inclusion in the database. The BIO-NMD database is now under construction and should be ready for M18 as planned.
Concomitantly, the INSERM team has started the development of various prediction tools that will be able to predict the pathogenicity of mutations found in next generation sequencing projects. The first tool, the Human Splicing Sequences Finder tool (HSF®, http://www.umd.be/HSF/), is able to predict the impact of any given mutation on splicing signals (splices sites, branch points, auxiliary sequences) and could now be queried by web-services. The second tool, UMD-Predictor®, is under development and will be able to predict the pathogenicity of substitutions of any given transcript. This tool should also be available for M18.


University of Milan (UNIMI)
Leader: Cecilia Gelfi

Summary: The University of Milan will be mostly involved with biomarker discovery by studies on patient cells, muscle tissues and body fluids as well as biomarker discovery and validation in animal models.

Update: To better understand the consequences of collagen VI mutations UNIMI have focused attention on studies of mice deficient in the protein. This will provide new information about the effect that different protein expression has on the animal model.
Proteome analysis of three different muscles in Col VI-/- animal model have indicated that muscles are differentially influenced by collagen VI absence. The identification of proteins differentially expressed in diaphragm, tibialis and gastrocnemious indicated that cytoskeletal, stress response proteins and metabolism compartments are predominantly involved. Diaphragm is characterized by a general down regulation, whereas tibialis and gastrocnemious show a peculiar protein dysregulation particularly of metabolic proteins.

Royal Institute of Technology Stockholm (KTH)
Leader: Mathias Uhlen

Summary: The Royal Institute of Technology (KTH) is involved in checking that biomarkers discovered during the BIO-NMD project are specific to the muscular dystrophies under study. This will involve comparing samples from patients at different stages of the diseases and under different treatment programmes with healthy controls and with patients with unrelated NMDs

Update: On the 15th of November KTH launched a new version (v7.0) of the Protein Atlas. The portal has been designed to serve not only as a data storage and visualization interface, but also to provide a knowledge platform for protein characterization. The content includes protein expression of 10,100 genes determined by antibody-based proteomics. Proteins are characterised in terms of expression in normal and diseased tissue, subcellular localization and size. Together with the portal, a new set of antibodies will be made available to WP4 for biomarker validation.

 A proof-of-concept study is being performed on samples provided by the consortium and with antibodies targeting a panel of biomarker candidates suggested by BIO-NMD partners. The sample collection includes 48 samples collected from DMD patients and healthy people.

Discussions with BIO-NMD partners are still ongoing for obtaining valuable patient material since larger numbers of the samples are preferred to ensure that the next analysis will offer meaningful results.

Members of Prof. Mathias Uhlen's group involved in the project are Dr. Cristina Al-Khalili Szigyarto, Dr. Peter Nilsson, Dr. Jochen Schwenk and PhD student Burcu Ayoglu.


Ariadne Genomics Inc (ARIADNE)
Leader: Nikolai Daraselia

Summary: Ariadne's role within BIO-NMD is to develop a literature-derived biological knowledge base focusing on Neuromuscular Diseases (NMD).

Update: Ariadne has developed the first prototype of a knowledgebase which focuses on neuromuscular diseases for the BIO-NMD project. It uses software tools and information extraction technology to gather relevant knowledge from the literature and link it into a network of relationships between genes, proteins, cellular processes, pathways and metabolites all relevant in NMDs.

This network was then used by Ariadne's experts to construct mechanistic models for various NMDs and to analyse genomic data in order to come up with a list of possible (candidate) biomarkers for the NMDs. These biomarkers can now be validated by other partners in the project to make sure that they are clinically relevant.

An IT infrastructure has been agreed upon which will give the BIO-NMD partners access to this prototype knowledgebase. Partners will be able to use the software to generate new hypotheses, analyze their own experimental data and validate biomarkers. Ariadne will provide a one-day training session on the use of Ariadne Pathway Studio® and MedScan Reader® software at the next BIO-NMD meeting (Dec 1st 2010, Montpellier, France).


AppliedBiosystems (AB)
Leader: Simone Guenther

Summary: AppliedBiosystems will provide technological support for methodological development using their SOLiD Next Generation Sequencing technology. The methods will allow analysis of BIO-NMD samples.

Update: One of AB’s roles is to develop methods for next generation sequencing for applications which are of critical importance to the whole BIO-NMD project, for example whole genome sequencing, targeted re-sequencing and whole transcriptome analysis. To this end, AB has started working with Professor Ferlini’s laboratory at the University of Ferrara to set up and optimize a method for the targeted re-sequencing of a panel of genes related to NMD.
For this purpose the University of Ferrara provided AB with a list of approximately 230 candidate genes (list generated by ARIADNE and including all genes in pathways related to both DMD and COL6) for the design of the enrichment array which is currently being manufactured. In the meantime, the team at the University of Ferrara is collecting a representative set of samples and will provide AB with the DNA for targeted re-sequencing experiments. For data analysis the SOLiD bioinformatics pipelines will be adapted and results will then be cross-validated with capillary electrophoresis based Sanger sequencing to define the best conditions for optimal sensitivity and specificity of the assay. This work will be ongoing until February/March of 2011.


Projets et Reseaux de Recherche (P2R/ACIES)
Leader: Loïc Courtot

Summary: ACIES/P2R will act as support to the project coordinator in WP
10 - Strategic and operational management. More specifically, ACIES/P2R will accompany the project coordinator in monitoring, evaluating, consolidating and submitting the project deliverables and periodic reports as well as monitoring the project progress in terms of quality, costs, schedule and risks, in accordance with EU regulations and the grant agreement.


Update: During the last 6 months ACIES/P2R as the partner responsible for operational management, organised the M6 meeting (July 7/8, London) along with the project coordinator (UNIFE). The M6 meeting was an opportunity to present the work progress and to the network and brainstorm. Industry was also represented. The objectives and outcomes of the project were presented, and they could interact with the researchers. For ACIES/P2R, it was also the chance to present the status of the project from a financial and reporting perspective.

The management team has also monitored the delivery of the outcomes and related reports/deliverables, the follow-up of the Industry meeting and supported the BIO-NMD partners in their work on a daily basis.

ACIES/P2R is still working on some legal issues (such as the Consortium
Agreement) and organisational matters such as the upcoming meeting in
Montpellier (December 1/2/3, 2010).


Conclusion: Targets have been met by the BIO-NMD partners with all deliverables either being achieved or in place to be achieved by the end of M12 (November 2010).

Many thanks are due to partners for their responses to my requests for input.

 

 

 




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