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Project update May 2011 - Full Version


University of Ferrara (UNIFE)
Leader: Alessandra Ferlini

Summary: The University of Ferrara is coordinating the BIO-NMD project. Their tasks are focused on discovering biomarkers by looking at DNA and RNA (genomic biomarkers) using both OMIC and standard genetic procedures. The team is also involved in proteomic studies by searching for circulating/low invasive cells/tissues as biomarkers as well as in providing patients’ plasma/tissues for identifying proteomic biomarkers. UNIFE is also involved in bioinformatics tools validation.

UNIFE is leading the Intellectual Property Committee (IPC).

Update:  The complete analysis of samples by proteomics, transcriptomics and genomics techniques requires the exchange of biomaterials and data between the partners of the BIO-NMD project. We have both received and delivered to KTH partner samples regarding plasma and serum of DMD and BMD obtained from our Italian sites as well from the Telethon Italy biobanks. KTH will perform protein array studies to identify peculiar set of proteins in these two phenotypes.

The DNA samples from patients with an atypical response to corticosteroids and patients with COLVI myopathies treated with cyclosporine A are under analysis by Applied Biosystems using capturing systems and high throughput SOLiD platform. The genes represented in the enriched customised array have been previously selected by the ARIADNE software, MedScan. The DNA sequencing (SNPs characterisation) by SOLiD has already been performed on the first samples. For the DMD patients with an atypical response to corticosteroids, RNA has been prepared and will be sent to UCL for miR analysis, whilst muscle tissue has been sent to UNIMI for proteomics analysis.

Following our expression profiling on both wild type and COLVIA1 KO mice we obtained a great variety of up/down regulated transcripts especially those classes related to aging, apotptosis and fibrosis and we delivered these data to INSERM and ARIADNE for bioinformatics analysis in order to build up interaction pathways/genes’ groups. This will allow us to identify subgroups of genes with modified expression which can then be further validated in patients.

We have performed RNAseq (Illumina) on patients (RNA from skeletal muscle) both before and after cyclosporine A treatment in order to identify altered RNA populations/SNPs. These data (completed) have been sent to INSERM and ARIADNE to finalise the bioinformatics analysis.

Both whole Exome Sequencing and RNAseq were performed in a female symptomatic carrier without mutations identified in the DMD gene while whole exome sequencing was carried out in a family with dominant inheritance of Bethlem myopathy, but with no mutations identified in the three COLVI genes. In these samples we aim to identify new causative genes (gene discovery/genetic biomarkers) and novel mechanisms explaining the DMD and BM phenotypes (pathophysiological biomarkers, gene modifiers).

Moreover, ChIP on Chip analysis in the DMD locus was performed by means of a new custom microarray covering the DMD gene locus. We evaluated the distribution of RNA Polymerase II and its major phosphorylation of CTD domain (Ser2 and Ser5) in the DMD gene. Furthermore we characterized the chromatin context (acethy-H3, H3K36 3Me, H3K4 2Me) in which Polymerase II works. The principal aims of this project are: understanding the transcriptional dynamic of DMD genes and the identification of cis and trans elements crucial to dystrophin regulation (pathophysiological biomarkers).

We developed with AB a new diagnostic tool (Flu-Omic cards)  able to analyse all the DMD transcript at once by using only 200 ng of total RNA from muscle and able to identify deletions/duplication, small and splicing mutationstherefore representing a suitable diagnostic tool (manuscript in preparation). The method also allows determination of RNA quantification by detecting decay timing during transcription.

Finally, based on the novel ncRNAs we previously identified as originating from the dystrophin locus, we are proceeding in depleting these RNAs using constructs containing antisenses, in order to establish their effect on dystrophin isoforms expression regulation/role in controlling dystrophin transcription (paper submitted).

Monocytes-derived macrophages produce and secrete collagen VI. We tested its expression in peripheral blood macrophages from patients with collagen VI-related myopathies and compared with muscle biopsy. RNA and protein studies were performed in blood macrophages from 5 patients previously diagnosed with either Ullrich congenital muscular dystrophy (UCMD) or Bethlem myopathy (BM). The full spectrum of possible genotypes was considered, including both dominant and recessive UCMD and BM cases. In the dominant BM patient, no collagen VI alterations were detectable in macrophages or muscle biopsy. In the remaining patients, the protein defect caused by the selected mutations, as well as the transcriptional abnormalities, were readily detectable in macrophages at levels comparable to those observed in muscle biopsy samples and cultured skin fibroblasts. Our data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing muscle/skin biopsies in the diagnosis and monitoring of collagen VI-related myopathies. These data have been recently published (Gualandi, Muscle and Nerve 2011).

Other studies demonstrate that normal epidermal melanocytes express dystrophin and are absent in the epidermis of DMD patients, while dystroglycans and BM components were normally expressed suggesting that epidermal dystrophin function is impaired in DMD patients. Since melanocytes can be easily obtained by conventional skin biopsy, they may represent a feasible and reliable cellular model for studying and monitoring dystrophinopathies.

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

Summary: Research at the LUMC primarily focuses on the assessment of disease severity in DMD, BMD and Col6 patients using protein biomarkers in blood. To this end, three approaches were taken: a targeted approach using ELISA and two non-targeted mass spectrometry (MS) based proteomics approaches.

Update: Targeted approach - Multiplex immunoassay technology was applied to examine 17 cytokines in sera from 3 patient groups: DMD (n=63), BMD (n=11) and healthy controls (n=20 children and adults). Significant differences in several mean chemokine levels were found but the variability in DMD patients appeared to be high.

MS based approaches - The focus of MS based approaches was geared towards increasing the number of potential protein biomarkers that can be identified in serum. Serum samples from 8 age matched DMD patients and control patients were collected and 2 pools of 4 patients were analyzed for both DMD and controls. In the first approach, Proteominer was used to equalize protein concentrations and to improve detection of medium to low abundant proteins. Serum proteins were then separated by means of gel electrophoresis, digested with both trypsin and Lys-N and analyzed using LC-ESI-MS/MS.

The second line of research concerns a gel-free approach applying 2D-HPLC separation (weak anion exchange and reverse phase separation) of intact serum proteins prior to tryptic digestion and LC-MALDI-TOF-MS. This method is now at the last stage of testing and will be applied to the above sample set.

For both approaches, peptide identification was carried out by matching fragmentation patterns against the Swissprot database using MASCOT. Analyses are ongoing and peptide lists for biomarker discovery are being generated.

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 - UNEW has successfully collected and exported large numbers of biomaterials (DNA, plasma, serum and muscle biopsies) to LUMC, KTH, UNIFE and UCL and provided comprehensive clinical data on each patient.  UNEW has also shared these clinical data with WP6 (Bioinformatics) as an example of what should be included in the BIO-NMD database.

By reaching out to EuroBioBank partners and in collaboration with UNIFE, an additional 42 muscle DMD and BMD muscle biopsies have now been retrieved. Transfer of muscle biopsies between UCL and UNIFE is planned over the next few weeks. Further sample collection and exchange are anticipated over the next few months to ensure good progress of the BIO-NMD project.  Consistent communication between 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 again to EuroBioBank and patients’ registries through the TREAT-NMD platform will be a possibility.

WP8 and 9 – During the last 6 months, UNEW has been working to establish links with various events and has coordinated the submission of a project abstract to WMS as well as presentations by partners to large expos. This continues and advice is sought from all partners as to the most appropriate events and meetings on which to focus.

Work has also begun on gathering information from all partners on their own dissemination activities relevant to BIO-NMD. Alongside this has been the establishment of the IPC and the guidelines for procedures to be followed in order to protect partners’ IP. These will be published in the near future once approval is confirmed by Novamen. Dissemination activity will be collated by UNEW and submitted to the EC as required. It will also form part of the plan for exploitation and future dissemination which is currently in progress.

The other tasks for these WPs have also continued – the PEB has met; the website has been maintained and updated; dialogue with the PAC has been happening and a representative will attend the Freiburg meeting; the next BIO-NMD newsletter is in progress.

University of Padova (UNIPD)
Leader: Paolo Bonaldo

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

Update: In the last 6 months, UNIPD has continued the study of self-degradation pathways (autophagy) in mice with COL6 mutations (Col6a1 null) and in Bethlem and Ullrich patients. These studies allowed the elucidation of molecular mechanisms underlying the impaired activation of autophagy in muscles of mice and patients. Defects in the activation of the autophagic process are responsible for the accumulation of altered organelles and death of muscle cells, causing muscle wasting and weakness. We found that forced reactivation of the autophagic process in mice with COL6 mutations by genetic, nutritional and pharmacological approaches is able to restore muscle cell survival and ameliorate the dystrophic phenotype. These findings allowed the identification of several novel candidate biomarkers for COL6 diseases.

During the last months, UNIPD has also undertaken studies aimed at investigating the regenerative potential of muscles in mice with COL6 mutations, both in normal conditions and in response to injury. We found that collagen VI is abundantly deposited in the extracellular matrix surrounding newly forming muscle cells during the initial phases of regeneration, suggesting a role for collagen VI in the process. In agreement with this, mice with COL6 mutations display reduced and delayed regeneration of muscle tissue. We are currently investigating the molecular mechanisms involved in the regenerative defects, with the aim of identifying additional 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:  In order to find candidate biomarkers and disease modifying variants for Duchenne Muscular Dystrophy (DMD) UCL is performing whole exome sequencing in two groups of DMD patients. The first group comprises five DMD patients on steroid treatment who lost ambulation before the age of 8.5 years and five DMD patients who lost ambulation after the age of 12 years. The second group consists of eight DMD patients with long survival (alive after 28 years) and a control group of DMD patients with early death (below the age of 18).
Quality control steps were carried out and only the DNA samples passing QC were included in the subsequent analysis. The whole exome sequencing was performed using exon enrichment kit (Sure Select 50 Mb XT) which covers 99.86% of the human protein coding regions annotated in NCBI Consensus CDS database. The prepared libraries were sequenced on Illumina GAIIx using 76 bp pair-end reads. The whole exome sequencing of the patients in the groups "Age at loss of ambulation" has been completed and the analysis of the data are ongoing.

In the last 6 months UCL has been collecting samples from DMD, BMD and Col6 patients and currently has collected 40 plasma and serum samples. In respect of sample distribution, UCL has sent muscle tissue from Col6 and DMD patients to UNIFE and serum and plasma samples to LUMC and KTH.

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: In the last BIO-NMD meeting (Leiden, 2-3 Feb 2011) UNIROMA explained to members of Consortium some EMA (European Medicine Agency) guidelines, that are of interest in the context of the BIO-NMD project.  In particular, specific information has been provided on genomic data and sample coding categories: guideline ICH15-EMEA/CHMP/ICH/437986. Furthermore, UNIROMA introduced the role and objectives of a multidisciplinary group within EMA - the Scientific Advice Working Party (SAWP), a standing working party with the remit of providing scientific advice and protocol assistance to applicants to facilitate an informal sharing of scientific and technical information between applicants and regulators. UNIROMA also provided some information on a document published in 2007 that explains to applicants how the regulatory agencies such as the FDA (Food and Drugs Administration in the US) and EMA, will process requests via the joint FDA/EMA voluntary genomic data submission (VGDS) briefing meetings.

In the last few months UNIROMA has uploaded these guidelines along with those discussed at the M6 meeting (London, 8-9 July 2010) to the BIO-NMD intranet ( UNIROMA is also continuing to follow the drafting of new documents (in progress) by the EMA.

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: As WP6 leader, the INSERM partner has been involved in the various aspects of the Bioinformatics including: a) development of bioinformatics tools for integrating and harmonizing the out flow of data and b) development and implementation of in silico tools for identifying functional pathways and potential Biomarkers.

During the first 18 months, INSERM had:

  • Ensured the design of the BIO-NMD database
  • In a first step it combined data from various projects and from the literature in order to submit a questionnaire about the database content and interface to all partners. After receiving feedback, partners have validated all options and the BIO-NMD database was designed.
  • Thanks to data received from LUMC and UNEW partners, the phenotype module has been implemented and is currently under validation. The other modules will soon be validated thanks to the submission of –omics data by all partners.
  • Developed a new system called UMD-HTS® that can handle the large sets of data generated by NGS and perform pathogenicity predictions for each SNP (Single Nucleotide Polymorphism). 
  • The INSERM partner collected from the Ensembl database (EBI) data from 32,734 genes, 62,031 transcripts, 524,515 exons and 462,484 introns. 
  • The coding sequences were automatically reconstructed from the crude dataset in order to generate all possible nucleotide substitutions from exonic sequences. At this step, an unexpected process of errors-correction was necessary as few hundreds of transcripts had wrong annotations. 
  • All substitutions resulting in stop codons were excluded, as their pathogenicity is usually obvious, leading to a total of 179,873,844 substitutions. 
  • In order to perform predictions using the UMD-Predictor algorithm, data were collected at various levels including conservation, physicochemical properties and impact on splicing signals. This led to a total of 3,957,224,568 annotations.
  • The UMD-HTS® system contains 24 satellite databases (one per chromosome) and a central system. A web interface was designed to query the system and because of the complexity of the parallel computing a long validation process was set-up.
  • Together with the UNIMED partner who provided data from various NGS experiments (5 patients), we evaluated the system as well as predictions’ quality in a blind experiment.

Concomitantly to WP6, the INSERM was also involved in WP4. Thus it contributed to the evaluation of an IL6R SNP biomarker. From the database of DMD and BMD patients available in the Laboratory of Molecular Genetics / INSERM U827, patients with required criteria (identified mutation in the DMD gene, age at loss of ambulation known and data on treatment by steroids available) have been included to the SNP validation study led by Peter 't Hoen (LUMC). From our database a total of 115 patients were initially selected and analysis of the SNP in the IL6R gene could be achieved in 110 patients. The results of genotyping have been integrated in the whole SNP validation study.

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: Human samples - UNIMI recently received from UNIFE, N.6 and N.3 DMD muscle samples treated and untreated with steroids. Of these, 5 were ambulant and 4 were non ambulant.
They also received many muscle samples (DMD and BMD) for proteomic studies.
Animal models - The effects of CsA treatment and ageing were assessed in the same muscles previously analysed to determine the proteomic signature of the Col VI knock-out model.  A set of molecules, as putative targets of CsA treatment, were identified. A partial recovery of deregulated proteins, more evident in gastrocnemious muscle, was observed according to muscle function recovery. These molecules could be considered as putative biomarkers for the follow up of CsA treatment and possibly validated in patients.

The effects of ageing were monitored on the same model. The ageing process indicates that different muscles react differently to Col VI absence and those differences are maintained through the course of ageing. The most affected muscle is the gastrocnemious in which energy balance, contractile and structural proteins are deregulated compared to that in young animals.

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

Summary: The Royal Institute of Technology (KTH) is involved in the screening, discovery and verification of biomarkers within the BIO-NMD project. This involves 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: During the first part of 2011, KTH has been engaged in selection and validation of antibodies for serum and protein plasma profiling.  In collaboration with other partners a list of genes that are potential biomarkers was compiled. Antibodies against NMD biomarker candidates were selected from the repository of the Protein Atlas program. The repository was searched for antibodies that target selected genes and that meet validation criteria for specificity. Only validated antibodies that have passed all quality validation tests - protein arrays, Western Blot and immunohistochemical staining were considered. In the first trial we selected 128 validated antibodies that have been suggested by two or more partners. The aim now is to select additional antibodies (up to 384) and generate a more complete list.
In parallel, serum and plasma samples have been received from Newcastle (UNEW) from patients diagnosed with DMD and BMD as well as from female carriers. Samples from UNIFE and UCL will be received in the near future. 

Ariadne Genomics Inc (ARIADNE)
Leader: Nikolai Daraselia

Summary: Ariadne’s role is to develop an integrative platform to analyse and mine the flow of generated data in the context of NMD. Ariadne MedScan® technology was used to build a literature-derived biological knowledge base focusing on neuromuscular diseases. Ariadne scientists used the environment to build mechanistic model pathways relevant for NMDs and initiated the analysis of experimental data for the identification and prioritisation of candidate biomarkers. Ariadne will further help with discovery and validation of different types of candidate biomarkers from disease diagnostic biomarkers to treatment efficacy biomarkers for personalised medicine.
Update: During the last 6 months, the Ariadne R&D team:

  • Completed the analysis of publicly available gene expression data to come with a short list of surrogate biomarkers for DMD progression. The candidate biomarkers identified by this analysis workflow have been critically analyzed and validated using literature resources. A panel of surrogate candidate biomarkers that show significant statistical correlation with the 12 month DMD progression and disease severity have been defined.  
  • Integrated feedback from partners to improve the BIO-NMD knowledgebase prototype.
  • Increased the collection of mechanistic model pathways and improved the existing ones
  • Collected DMD and Col6 candidate biomarkers from partners. Provided a list of best candidate biomarkers for WP3 activities and the development of a bead array immunoassay to screen potential biomarker in serum and plasma samples from DMD patients
  • Initiated the analysis of partners’ experimental data for WP2 activities

Also, Ariadne technical support staff:

  • Put in place the IT infrastructure to host the BIO-NMD knowledgebase on a server. 
  • Coordinated deployment of Pathway Studio and MedScan software at partners’ sites and the access to the BIO-NMD knowledgebase for end-users.
  • Provided an introductory one-day training course on Pathway Studio and MedScan Reader to partners.

AppliedBiosystems (AB)
Leader: Simone Guenther

Summary: Applied Biosystems, part of Life Technologies 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: In the role of next generation sequencing methods developer for applications which are of critical importance for Biomarker discovery for NMD, AB 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.

230 candidate genes were considered for the development of the first panel. These genes were identified using Ariadne analysis tool as genes correlated to the pathways of the two main NMD genes (DMD and COL6) and validated with some expert advice. We designed a panel of probes for enrichment of these genes, which was used to capture the exons of 20 samples provided by the Professor Ferlini’s group.

6 of these 20 samples have already been processed through the SOLiD sequencing workflow to test for the performance of the enrichment kit and optimize the bioinformatics pipeline. These first samples have been sequenced at redundant depth so to allow simulations of decreased coverage to verify the robustness of variant calling.

A dedicated Bioinformatic specialist has been hired for the project and will follow all future method development from an analysis point of view. Testing the method includes verification of the percentage of exon sequence we manage to sequence at a depth which enables an adequately accurate call of variants (SNPs and in-dels), sensitivity and specificity of the method, simulations to identify best conditions for future experiments. We expect data analysis for the initial subset of samples to be finished by the end of May 2011 and a tested protocol to be immediately available for subsequent analysis of larger series of samples, including the rest of 14 samples already provided to AB.

Novamen (formerly P2R/ACIES)
Leader: Roseline Favresse

Summary: Novamen 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 P2R/NOVAMEN as the partner responsible for operational management, organised the M12 meeting in Montpellier (1-2 December 2010). Due to the low attendance of partners to this meeting because of unforeseen climatic events all over Europe, the M12 meeting was reconvened in Leiden, the Netherlands (2-3 February 2011) at M15, right after the Governing Board of the EU-funded FP6 TREAT-NMD project. During the last 6 month period, NOVAMEN has also been involved in the planning of the M18 meeting to take place in Freiburg, Germany (8-9 June 2011).

As of May 2011, the BIO-NMD project is at mid-term and entering quite a challenging period as far as WP10 is concerned. At M18, 22 deliverables have to be released to the EC (i.e. one third of the total number of the project deliverables) as well as the first official reporting related to the M1-M18 period. Bi-annual meetings remain a true opportunity for partners to present the work progress and to brainstorm.

Over the period, the management team has also monitored the delivery of the outcomes and related reports/deliverables and supported the BIO-NMD partners in their work on a daily basis. As the project partners will report financially for the first time to the EC, increased support is provided to partners.

The Management partner will finalise the consortium agreement in May. Organisational matters have been followed up, such as the organisation of the upcoming M18 meeting, in liaison with the FP6 TREAT-NMD consortium and the DG SANCO Project CARE-NMD which will convene their meetings the same week in Freiburg.



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