Gert-Jan B. van Ommen

Biological resources (cells, tissues, bodily fluids or biomolecules) are considered essential raw material for the advancement of health-related biotechnology, for research and development in life sciences, and for ultimately improving human health. Stored in local biobanks, access to the human biological samples and related medical data for transnational research is often limited, in particular for the international life science industry. The recently established pan-European Biobanking and BioMolecular resources Research Infrastructure-European Research Infrastructure Consortium (BBMRI-ERIC) aims to improve accessibility and interoperability between academic and industrial parties to benefit personalized medicine, disease prevention to promote development of new diagnostics, devices and medicines. BBMRI-ERIC is developing the concept of Expert Centre as public–private partnerships in the precompetitive, not-for-profit field to provide a new structure to perform research projects that would face difficulties under currently established models of academic–industry collaboration. By definition, Expert Centres are key intermediaries between public and private sectors performing the analysis of biological samples under internationally standardized conditions. This paper presents the rationale behind the Expert Centres and illustrates the novel concept with model examples.

Background: Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression. Results: Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes. Conclusions: The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

Background Huntington's disease (HD) is a devastating disease for which currently no therapy is available. It is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD gene which results in an expansion of polyglutamines at the N terminal end of the huntingtin (htt) protein and the accumulation of cytoplasmic and nuclear aggregates in neurons. The polyglutamine expansion results in a toxic gain of function for the huntingtin protein and plays a central role in the disease. The size of this expansion has a direct link to the aggregation proneness as well as the severity of pathological and clinical features. Aims The aim of our study is reducing mutant htt transcript and protein levels by targeting its prolonged CAG repeat in the HD gene and thereby inhibiting all downstream toxic effects. Methods Patient derived fibroblasts and lymphoblasts were used to transfect fully modified 2'O methyl phosphorothioate antisense oligonucleotides (AONs), designed to target the expanded CAG repeat in HD. Various AON concentrations and CUG lengths were applied. Htt transcript levels were measured by RT-PCR and qRT-PCR and protein levels were measured by Western blot and time resolved FRET. Results We show that an AON that recognises seven consecutive CAG trinucleotides reduces both mutant htt transcript and protein levels in patient derived cells. In the human genome there are numerous proteins that contain polyQ tracts and these are usually encoded by a combination of CAG and CAA triplets. Those CAG enclosing transcripts were found to be unaffected by the (CUG)₇ AON treatment. Conclusions Here we make use of AONs targeting the CAG repeat as a therapeutic strategy to effectively reduce both htt transcript and protein levels in patient derived HD cells. Other endogenous CAG enclosing transcripts were found to be unaffected by AON treatment, making the (CUG)₇ AON a promising therapeutic tool to specifically reduce mutant htt in HD.

Genes that play an important role in tumorigenesis are expected to show association between DNA copy number and RNA expression. Optimal power to find such associations can only be achieved if analysing copy number and gene expression jointly. Furthermore, some copy number changes extend over larger chromosomal regions affecting the expression levels of multiple resident genes.

Background: Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge. Results: HRMA results nicely matched those obtained with ELISA and compared favourably to DNA fingerprinting of restriction digested clone insert-PCR. DNA sequence analysis confirmed that HRMA-clustered clones contained identical inserts. Conclusion: Using HRMA, analysis of up to 384 samples can be done simultaneously and will take approximately 30 minutes. Clustering of clones can be largely automated using the system's software within 2 hours. Applied to the analysis of clones obtained after phage display antibody selection, HRMA facilitated a quick overview of the overall success as well as the identification of identical clones. Our approach can be used to characterize any clone set prior to sequencing, thereby reducing sequencing costs significantly.

The specific skipping of an exon, induced by antisense oligonucleotides (AON) during splicing, has shown to be a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients. As different mutations require skipping of different exons, this approach is mutation dependent. The skipping of an entire stretch of exons (e.g. exons 45 to 55) has recently been suggested as an approach applicable to larger groups of patients. However, this multiexon skipping approach is technically challenging. The levels of intended multiexon skips are typically low and highly variable, and may be dependent on the order of intron removal. We hypothesized that the splicing order might favor the induction of multiexon 45–55 skipping.

WikiProteins enables community annotation in a Wiki-based system. Extracts of major data sources have been fused into an editable environment that links out to the original sources. Data from community edits create automatic copies of the original data. Semantic technology captures concepts co-occurring in one sentence and thus potential factual statements. In addition, indirect associations between concepts have been calculated. We call on a 'million minds' to annotate a 'million concepts' and to collect facts from the literature with the reward of collaborative knowledge discovery. The system is available for beta testing at http://www.wikiprofessional.org.