News & updates

M6 Full Version

Project Update May 2010 - Full Version

Introduction: The BIO-NMD project is a collaboration of 12 European partners working together towards the following outcomes:

• Identification of preliminary and exploratory biomarkers
• Validation of exploratory biomarkers in cells and animal models
• Validation of exploratory biomarkers in human body fluids
• Translation of both diagnostic and pathophysiological data to pre-clinical research
• Definition of a specific biosignature for the NMDs in the study
• Validation of identified biomarkers following the EU rules for regulation and qualification

The project is currently in its 6th month and much progress has been made. This report summarises the activities of the partners during that time. It is intended only as a brief update to complement ongoing discussions and communication between partners.


University of Ferrara (UNIFE)
Leader: Alessandra Ferlini

Summary: The University of Ferrara is coordinating the BIO-NMD project. In addition, UNIFE leads WP 2 - Genomic biomarkers discovery by genome-wide analyses of DNA and RNA. The team is also involved in providing patients' samples, performing high-throughput genomic analysis and transcription analysis and participating in biomarker validation.

Update: During these first months UNIFE has:
• finalised and delivered the BIO-NMD web site
• got approval from the Local Ethics Committees together with licences for animal experiments
• finalised the creation of a patient friendly section on the project website with updated information about the progress of BIO-NMD
• set-up the translational task force (composed of F.Muntoni, H. Lochmueller, L.Merlini, P.t’Hoen, V.Straub, N. Levy and A. Ferlini), for selecting a very limited number of patients to be studied by OMIC approaches. The task force is working on identifying categories and criteria to define the list of patients and related biomaterial sources.
• built-up a list of candidate genes for both DMD and COL6 myopathies to be analysed using the ARIADNE medscan software in order to identify common pathways to be further explored by specific assays and/or integrated with the OMIC work.

At the London meeting (8-9 July 2010, hosted by Francesco Muntoni) UNIFE has organised a dedicated session for industry in which the project impact and possible outcomes will be presented and described. Links for future collaborations will also be identified.


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

Summary: LUMC is the leader for WP 3 - Proteomic biomarkers discovery by studies on patient cells, muscle tissues and body fluids, and WP 4 - Exploratory biomarkers validation in humans. This is based on the partner's previous expertise with biomarker identification in NMD mouse models. LUMC is also involved as a partner in other work packages.

Update: LUMC has screened the serum of Duchenne patients for candidate protein biomarkers with ELISA – an immunosorbent assay that is based on antibody detection. They have found two proteins with elevated levels in DMD patients compared to healthy age-matched controls.


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

Summary: The Institute for Human Genetics at Newcastle University is involved in three work packages for the project: WP 1 - Patients' material organisation, circulation and managing of novel patients' fluids collection; WP 8 - Social and ethical aspects; and WP 9 - Exploitation and dissemination.

Newcastle University has its own biobank containing human material for dystrophinopathies and collagen VI disorders as well as playing a key role in EuroBioBank. This enables them to effectively lead WP1.

As the coordinators of the TREAT-NMD network of excellence, Newcastle University is also able to draw on existing networks and dissemination tools to enable the team to lead WPs 8 and 9.

Update: Two new members of Newcastle’s BIO-NMD team were recruited and began working on the project in April. Cathy Turner has been working on WPs 8 and 9 whilst Amina Chaouch is working closely with Hanns Lochmueller on WP1.

In recent months, the patient-friendly part of the project’s website has been created assisted by much discussion with and input from the PAC. Other parts of the website have been created or improved in order that it can be used as a valuable dissemination tool and discussion forum for BIO-NMD.

The project ethics board (PEB) has arranged its first meeting in Barcelona in June and a summary of the outcomes will be posted onto the website.

As part of its involvement with WP1 (Patients material organisation, circulation and managing of novel patients’ fluid collection), UNEW has been liaising with local and international repositories to ascertain biomaterial availability, how much more is needed and when.

They have also been working as part of the clinical task force (F.Muntoni, H. Lochmueller, L.Merlini, P.t’Hoen, V.Straub, N. Levy and A. Ferlini) in selecting clear clinical criteria for the various patient subgroups. In addition, UNEW has been discussing with partners the need for a standard protocol to adhere to when collecting samples across the different centres.


University of Padova (UNIPD)
Leader: Paolo Bonaldo

Summary: The University of Padova is the leader of WP 5 – Biomarker discovery and validation on animal models. The main tasks of WP5 are the analysis, validation and therapeutic exploitation of biomarkers in animal models.

The team has broad and well-proven expertise in studies in mice with a strong background on mouse models for collagen VI myopathies. Their main tasks will be the analysis, validation and therapeutic exploitation of biomarkers in animal models.

The University of Padova will also be involved in other work packages as a partner.

Update: During the first 6 months UNIPD has

• Performed pathophysiological and molecular studies on COL6 null (Col6a1 knockout) mice, to obtain novel insight into the pathomolecular mechanisms that may also have useful value as biomarkers.
• Started the phenotypic characterization of a new animal model for COL6 myopathies, generated by the UNIPD group. This is a transgenic mouse strain carrying inducible Col6a1 RNAi silencing, where COL6 synthesis can be turned on by doxycycline in an inducible manner. Interestingly, findings obtained with these mice show that different levels of COL6 knockdown can be recapitulated on the muscle phenotypic effects.
• Carried out experiments aimed at throwing light on the mechanisms responsible for muscle cell death.
• Subjected mouse models to training exercise and assessed muscle function and various known biomarkers (such as mitochondrial dysfunction, apoptosis and regeneration) 
• Built up a list of candidate genes for COL6 myopathies (including candidates detected by studies on mice in vivo, known interactors and binding partners for collagen VI, and functional candidates detected by in vitro studies), to be used by Ariadne for the generation of a knowledge base. 


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 discovery of both genetic and protein biomarkers that would indicate differences in disease progression and/or response to potential therapeutic agents.  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.

UCL Institute of Child Health will contribute mainly to WP 1 - Patients' material organisation, circulation and managing of novel patients' fluids collection; WP 2 - Genomic biomarkers discovery by genome-wide analyses of DNA and RNA; and WP 3 - Proteomic biomarkers discovery by studies on patient cells, muscle tissues and body fluids

Update:  Prof. Francesco Muntoni has been actively involved in the design of the study for biomarker selection, in particular the selection of the patient phenotypes.

UCL will host the BIO-Consortium Meeting in June 2010 in London.

UCL has recently recruited two full staff to work on the BIO-NMD Project:

Dr. Sebahattin Cirak as senior clinical research associate who is a paediatrician with longstanding research experience in genetics and clinical research in neuromuscular disorders.

Dr. Irina Zaharieva as postdoctoral research associate with expertise in association studies of complex disorders (Schizophrenia, ADHD) using SNP and CNV arrays.

Dr. Cirak has started the recruitment of patients for the study and Dr. Zaharieva has started to establish full RNA sequencing using next generation sequencing (Illumina GA2 Platform).


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

Summary: The university will be leader of WP 7 - Biomarkers regulation and qualification at the EMEA. The team will also address other WPs' work in the process requested for regulation and qualification of biomarkers. This will create a network between the different WPs, thus facilitating their relationship with EMA and assisting the applicants in the submission of data, in order to obtain the qualification of proposed biomarkers in a short time frame.

Update: Prof. Novelli’s team is following the work in progress of the European regulatory agency concerning the definition of new  rules, documents and guidelines about Genomic Biomarkers (GB) definition, and about application for qualification and regulation processes of  these biomarkers in diagnostic and drug development.

This is in order to facilitate and guide the future the work of the other partners in the BIO-NMD project who will identify new genomic biomarkers facilitating and guiding them to apply for GB regulation and qualification.


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

Summary: INSERM is the leader of WP 6 - Bioinformatic 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 will be 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 will be 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.
Concomitantly, the INSERM team has started the development of the UMD-Predictor® software that will be able to predict the pathogenicity of missense mutations found in next generation sequencing projects. It will be used in addition to other tools that are able to predict the impact of intronic mutations (Human Splicing Sequences Finder tool, HSSF®) as well as data from public databases such as dbSNP.


University of Milan (UNIMI)
Leader: Cecilia Gelfi

Summary: The University of Milan will be concerned with muscle proteome differential analysis by 2D-Dige in a selected number of patients and in animal models. They will be mostly involved with WP 3 - Proteomic biomarkers discovery by studies on patient cells, muscle tissues and body fluids; and WP 5 - Biomarker discovery and validation on animal models.

Update: To better understand the molecular consequences of Collagen VI absence UNIMI have focused attentions on the proteome comparative studies of wild type and Col6a1-/- mice. The systematic comparison of proteome performed on muscles with different disease progression will provide new information about how differential protein expression is related to the specific phenotypes displayed by their animal model.

Protein from diaphram, tibialis and FDB muscles of COL VI-/- and control animals were analysed by 2D-DIGE. Protein extracts (50µg) from each set type were labelled with 400 pmol Cy5 dye (CyDye, GE healthcare), while an internal standard, generated by pooling together an aliquot of all muscle samples, was labeled with Cy3 dye. Minimal labeling was performed according to manufacturer’s recommendations. Samples were separated on 24 cm, 3-10 non linear pH gradient IPG strips (GE Healthcare). Each sample was run in triplicate. Second dimension was carried out in 12% polyacrylamide gels. CyDye-labeled gels were scanned on a Typhoon imager (GE Healthcare). Differential protein expression was evaluated using 2D-DIGE on 3-10 NL  IPG strips. 2D gels were scanned on a Typhoon 9200 scanner and the digital images were analysed. After automatic spot detection, background subtraction, and volume normalization, approximately 4000 spots were detected. Differential analysis was performed by DeCyder software. Gel image analysis is now under construction.

Preliminary results of hierarchical cluster analysis of samples indicated that analysed muscles are differentially influenced by disease progression suggesting an active role of muscle tissue in the investigated model. Conclusion of the analysis is planned for the first week of July 2010.

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

Summary: The Royal Institute of Technology (KTH) is involved with WP 4 - Exploratory biomarkers validation in humans.

Update: KTH has been involved in setting up the Plasma Protein Profiling platform and implementation of the planned workflow.  A new LUMINEX instrument has been installed with higher multiplexing capability. The system enables analysis of 384 samples in one single run. The instrument has been tested and calibrated for initiation of the analysis. During the first 6 months we have also been in contact with BIO-NMD partners to discuss which type of patient samples (blood, serum or plasma) should be analyzed by proteomic methods, the amount required for the analysis and the number of the samples to ensure that the planned analysis will provide meaningful results.


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) and containing an NMD ontology; to build mechanistic models and pathways for NMD and to interpret ‘omics’ data generated by the consortium in the context of NMD knowledgebase and networks.

ARIADNE's main involvement is with WP 6 - Bioinformatic tools for identifying functional pathways, potential targets and data outflow integration

Update: ARIADNE have developed a detailed plan for building the first snapshot of an NMD knowledge base. They have been analyzing NMD-related literature in order to identify relevant concepts and make sure that MedScan dictionaries are optimized for generation of an NMD knowledge base.

They have also started building NMD-related pathways outlining the molecular mechanisms of various muscular dystrophies and are looking into public repositories of gene expression data to identify and import NMD-related datasets.

AppliedBiosystems (AB)
Leader: Simone Guenther

Summary: AppliedBiosystems will provide technology support for method development using their SOLiD Next Generation Sequencing technology. The methods will allow analysis of BIO-NMD samples for whole genome sequencing, targeted resequencing of specific candidate genes, whole transcriptome analysis and short RNA analysis.

AB's main involvement is with WP 2 - Genomic biomarkers discovery by genome-wide analyses of DNA and RNA

Update: AB have been working with Professor Ferlini’s laboratory at the University of Ferrara to set up and optimize a method for the targeted re-sequencing of 80 genes related to NMD.

Their role is in developing 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.

The plan is to get samples and a list of genes from the University of Ferrara and start work on the design of the enrichment array followed by re-sequencing and analysis. They will then cross validate the SOliD results with those obtained by capillary electrophoresis to define the best conditions for optimal sensitivity and specificity of the assay.


Projets et Reseaux de Recherche (ACIES/P2R)
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 Kick-Off Meeting for the project along with the project coordinator (UNIFE). It was an opportunity for the partners to present their duties and capabilities, and to network and brainstorm. For ACIES/P2R, it was also the chance to present the legal and financial aspects of this European project.

The management team has also monitored the delivery of the first outcomes and related reports, and supported the BIO-NMD partners in their work on a daily basis.

ACIES/P2R is currently working on some legal issues (such as the Consortium Agreement) and organisational matters such as the upcoming meeting in London (July 8/9 2010). Part of this event will also be opened to Industry.

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 M6 (May 2010) as shown in the table.

 No. Title of Deliverable   Responsible Due Date Achieved 
 D1.1  Report on biomaterial availability, collection and additional needs  UNEW  M6  On target
 D1.4  Report on biomaterial provision  UNEW  M6  M6
 D8.1  Creation of Project Ethics Board (PEB)  UNEW  M3  M3
 D8.2  Local Ethics Committee approval plus animal licences  UNEW  M3  M3
 D8.4  6 Monthly PEB meetings  UNEW  M6  M6
 D8.7  Creation of patient-friendly pages on website  UNEW  M5  M5
 D9.1  Creation of project website  UNEW  M3  M3
 D9.2  Dissemination of project report  UNEW  M6  M6
 D9.3  Creation of discussion forum on-line  UNEW  M6  M6
 D10.1  Project management manual  P2R  M3  M3

This report constitutes part of D9.2 and will be disseminated accordingly. Many thanks are due to partners for their responses to my requests for input.



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