Gjb van Ommen

Muscle regeneration in dystrophin-deficient mdx mice studied by gene expression profiling

BackgroundDuchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is lethal. In contrast, dystrophin-deficient mdx mice recover due to effective regeneration of affected muscle tissue. To characterize the molecular processes associated with regeneration, we compared gene expression levels in hindlimb muscle tissue of mdx and control mice at 9 timepoints, ranging from 1–20 weeks of age.ResultsOut of 7776 genes, 1735 were differentially expressed between mdx and control muscle at at least one timepoint (p < 0.05 after Bonferroni correction). We found that genes coding for components of the dystrophin-associated glycoprotein complex are generally downregulated in the mdx mouse. Based on functional characteristics such as membrane localization, signal transduction, and transcriptional activation, 166 differentially expressed genes with possible functions in regeneration were analyzed in more detail. The majority of these genes peak at the age of 8 weeks, where the regeneration activity is maximal. The following pathways are activated, as shown by upregulation of multiple members per signalling pathway: the Notch-Delta pathway that plays a role in the activation of satellite cells, and the Bmp15 and Neuregulin 3 signalling pathways that may regulate proliferation and differentiation of satellite cells. In DMD patients, only few of the identified regeneration-associated genes were found activated, indicating less efficient regeneration processes in humans.ConclusionBased on the observed expression profiles, we describe a model for muscle regeneration in mdx mice, which may provide new leads for development of DMD therapies based on the improvement of muscle regeneration efficacy.

The human genome project and the future of diagnostics, treatment, and prevention

The Human Genome Project, the mapping of our 30 00–50 000 genes and the sequencing of all of our DNA, will have major impact on biomedical research and the whole of therapeutic and preventive health care. The tracing of genetic diseases to their molecular causes is rapidly expanding diagnostic and preventive options. The increased insights into molecular pathways, gained from high-throughput ‘functional genomics’, using DNA-chip and protein-chip approaches and specially designed animal model systems, will open great prospects for pharmacological and genetic therapies. Powerful bioinformatics and biostatistics will further improve our pattern recognition and accelerate progress. A rapidly expanding area of high expectations is that of ‘pharmacogenomics’: the design of more effective drugs with lower toxicity through tailoring of drug treatment to individual, genetically determined differences in drug metabolism. Not only will this decrease the cost of health care through reduction of adverse drug reactions, but a better stratification of populations will also provide more statistical power farther upstream in drug trials. However, the optimal benefits from the current explosion of ‘data mining’ will only be realized when the basic data are made and kept publicly accessible, while at the same time safeguarding the protection of intellectual property arising from downstream inventions. This is one of the goals of HUGO, the international Human Genome Organization, established 13 years ago to assist coordination of data acquisition and exchange and societal implementation of the genome project. Additional points of attention in this historic endeavour are the prevention of stigmatization and discrimination and the safeguarding of a worldwide balance in the contribution by — and benefits to — different populations, while respecting the diversity in cultures and traditions.

Relative power and sample size analysis on gene expression profiling data

BackgroundWith the increasing number of expression profiling technologies, researchers today are confronted with choosing the technology that has sufficient power with minimal sample size, in order to reduce cost and time. These depend on data variability, partly determined by sample type, preparation and processing. Objective measures that help experimental design, given own pilot data, are thus fundamental.ResultsRelative power and sample size analysis were performed on two distinct data sets. The first set consisted of Affymetrix array data derived from a nutrigenomics experiment in which weak, intermediate and strong PPARα agonists were administered to wild-type and PPARα-null mice. Our analysis confirms the hierarchy of PPARα-activating compounds previously reported and the general idea that larger effect sizes positively contribute to the average power of the experiment. A simulation experiment was performed that mimicked the effect sizes seen in the first data set. The relative power was predicted but the estimates were slightly conservative. The second, more challenging, data set describes a microarray platform comparison study using hippocampal δ C-doublecortin-like kinase transgenic mice that were compared to wild-type mice, which was combined with results from Solexa/Illumina deep sequencing runs. As expected, the choice of technology greatly influences the performance of the experiment. Solexa/Illumina deep sequencing has the highest overall power followed by the microarray platforms Agilent and Affymetrix. Interestingly, Solexa/Illumina deep sequencing displays comparable power across all intensity ranges, in contrast with microarray platforms that have decreased power in the low intensity range due to background noise. This means that deep sequencing technology is especially more powerful in detecting differences in the low intensity range, compared to microarray platforms.ConclusionPower and sample size analysis based on pilot data give valuable information on the performance of the experiment and can thereby guide further decisions on experimental design. Solexa/Illumina deep sequencing is the technology of choice if interest lies in genes expressed in the low-intensity range. Researchers can get guidance on experimental design using our approach on their own pilot data implemented as a BioConductor package, SSPA http://bioconductor.org/packages/release/bioc/html/SSPA.html.

B07 Analysis of huntingtin protein fragments in post mortem human Huntington's disease brain tissue

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disease caused by elongation of a CAG-repeat within the first exon of the huntingtin gene. This mutation leads to a toxic gain-of-function of the huntingtin protein (htt). The exact mechanism of HD pathogenesis remains elusive, but it is thought that proteolytic cleavage of the mutant htt protein is an important step in HD pathogenesis. However, studies involving htt cleavage fragments in human brain tissue could be complicated by non-disease specific degradation of the htt protein during post-mortem delay. To elucidate the effects of post-mortem delay, we have conducted a study using human HD caudate nucleus tissue and human temporal lobe tissue as control with low initial post-mortem delays (3 and 1 h resp). To mimic post-mortem delay, specimens were brought to room-temperature and every 2 h samples were taken for a minimum of 8 h. Analysis of these samples was performed by Western-blotting using an antibody that recognises the first 17 amino acids. For both brain regions, the majority of fragments did not change between time points, apart from fragments at 52 kD and 70 kD which increased over time. Only in the HD caudate nucleus specimen, we observed several htt fragments between 80 to 100 kD that decreased over time. We conclude that post-mortem delay only has moderate effects. Next, we analysed interpersonal differences between the sensory/motor cortex and caudate nucleus region from nine different HD and control subjects. First results on Western blotting indicate that there are no striking differences between HD and controls for the sensory/motor cortex. For the HD caudate nucleus however, we observed an increase in protein fragments compared to the control samples. These initial results suggest that there is a regional variation in htt protein fragmentation in the human brain which may be related to pathogenesis.

P01 Antisense oligonucleotide mediated transcript reduction and modulation—the European approach to develop a therapy for Huntington disease

There has been a recent surge in research using antisense oligonucleotides (AONs) to reduce huntingtin transcript levels and thus huntingtin protein levels both in vitro an in vivo. This can be done in a non-allele specific manner by targeting both mutant and normal huntingtin transcripts but preferred would be an allele specific approach targeting mutant huntingtin transcripts through SNP-specific AONs or by use of triplet-repeat AONs complementary towards the (CAG)n expansion. Studies in our group and others have confirmed the feasibility of this approach. The disadvantage however is that there is a reduction in huntingtin protein levels and lowering huntingtin levels too much will cause unwanted side effects. Fortunately, AONs are a versatile tool that can also be exploited to induce inclusion or exclusion of target exons, thereby modulating the translated protein product. It is known that caspase 6 cleavage at aa 586 gives rise to a toxic N-terminal huntingtin fragment, and mutation of this site in the YAC128 mouse model (C6R-YAC128 by the group of Michael Hayden) provides protection from neuronal dysfunction and neurodegeneration. This (and other) caspase cleavage sites are encoded by exon 12 of the HTT gene. By skipping this exon with AONs, a shorter huntingtin protein will be formed lacking the caspase cleavage sites, without altering overall huntingtin protein levels. We propose a combinatorial AON approach for Huntington disease, making use of the transcript reducing properties of (CUG)7 AONs that preferentially targets mutant huntingtin transcripts, as well as the transcript modifying AONs that remove important caspase cleavage sites from the huntingtin protein.

B15 CTCF in Huntington's disease

Background Trinucleotide repeat expansion is the cause of at least 25 inherited neurological disorders, including Huntingtons disease (HD), fragile X mental retardation, and myotonic dystrophy (DM1). An interesting feature of repeat expansion mutations is that they are genetically unstable, meaning that increased disease severity and decreased age-of-onset are observed as the mutation is transmitted from parent to offspring. Previous studies on spinocerebellar ataxia 7 and DM1 have identified the cis-acting DNA element CTCF to be implicated in repeat expansion. CTCF is a regulatory factor implicated in genomic imprinting, chromatin remodelling, and DNA conformation change. Mutation or CpG methylation of CTCF binding sites promotes triplet repeat instability both in germ line and somatic tissue. Aim As binding sites for CTCF are associated with many highly unstable repeat loci, CTCF may also be involved in regulating genetic instability in HD. Methods We have in silico identified putative CTCF binding sites in the HTT locus. Confirmation of these CTCF binding sites has been achieved by electrophoretic mobility shift assays. Chromatin immunoprecipitation was performed with a CTCF antibody followed by deep sequencing (ChIP-Seq) in two HD patient fibroblasts and two wild-type control cell lines. Results Using electrophoretic mobility shift assays we have identified several CTCF binding sites in the HTT locus of which at least three CTCF binding sites are proximal to the CAG repeat. Analysis of the ChIP-Seq data showed 30 CTCF sites on the genome with differential CTCF binding between HD and control fibroblast cell lines. No differences in CTCF binding within the HTT locus were found between the HD and control cell lines. Conclusions No differential binding of CTCF to the HTT locus in fibroblasts that could suggest that CTCF can act as a modulator in genetic repeat instability in HD. Genome wide analysis and ChIP-Seq in brain tissue will determine if CTCF could be involved in HD pathogenesis.

F03 Whole blood SAGE digital gene expression profiling from Huntington's disease patients

Background The robustness and reproducibility of next generation sequencing technologies make them an excellent platform for gene expression profiling experiments. Despite the fact that the choice of tissue for RNA extraction and sequencing greatly depends on the clinical or research question, whole blood has become an increasingly popular choice. Especially in Huntingtons Disease where the pathology originates in a difficult to obtain samples tissue (brain) blood can show promising results. Aims We have compared 100 HD carriers with 50 age and sex matched controls and looked for transcriptomic biomarkers that can distinguish between the disease stage. Methods We have used the serial analysis of gene expression protocol with an NlaII restriction enzyme, adapted to yield products, for next generation sequencing analysis. By comparing the abundance of sequencing tags between patients and controls we can identify the differences between patients and controls. We have used the statistical platform R and two programs for differential expression analysis edgeR and Voom. For preliminary wet lab validation of our results we have performed immunostaining of the proteins of interest in brain lysates. Results We idenftify a set of 8 mRNA biomarkers that is differentially expressed between the late symptomatics and the control cases. Some of these biomarkers can be independently validated with another two programs for statistical analysis of gene expression data but also with wet lab techniques such as immunostaining from brain lysates between HD and control cases. Finally we are working and are interested in methods of identifying further relationships with this transcripts of interest and other transcripts but also working on validating these results in serum and on independent patient cohorts.

P03 Reducing toxic N-terminal huntingtin fragments in HD using exon skipping

Background Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis (Thornberry et al 1997). Huntingtin fragments within the striatum of human HD brains clearly differ from those of control brains (Mende-Mueller et al 2001), suggesting cleavage is disease specific. HD cell models with caspase-3 and caspase-6 resistant neuronal and non-neuronal cells showed reduced toxicity and were found to be less prone to aggregate formation (Wellington et al 2000). In YAC128 HD mice, expressing human genomic mutant huntingtin containing 128 glutamines, the HD phenotype was prevented by blocking caspase 6-specific cleavage by mutating the caspase-6 cleavage motif (Graham et al 2006; Pouladi et al 2009). This suggests that caspase-6 cleavage at position 586 is a key player in neuronal dysfunction and neurodegeneration. Aims For our study we make use of 2’O-methyl modified antisense oligonucleotides (AONs) with a phosphorothioate (PS) backbone to induce in-frame exon skipping in huntingtin of the exons in which the proteolytic cleavage motifs are located. Methods/techniques Patient derived fibroblast cells were transfected with AONs, RNA was isolated 1-day after transfection and skipping efficiency was determined. To assess the formation of a shorter, skipped protein, total protein lysates were obtained 3 days after transfection and shown by Western blot. A caspase-6 assay was used to determine the caspase-resistance of the truncated huntingtin protein. Results/outcome Patient derived fibroblast cells transfected with an AON binding to the 3’ part of exon 12, resulted in a partial skip of 135 base pairs. This partial exclusion of the 3’ part of huntingtin exon 12 can be explained by the existence of an cryptic 5’ splice site AG|GTCAG (Zhang et al 1998). The in-frame skip results in a slightly shorter huntingtin protein resistant to proteolytic cleavage at the caspase-6 cleavage site. In-frame multi-exon skipping of exon 12 and 13 from huntingtin pre-mRNA resulted in a shorter huntingtin protein resistant to proteolytic cleavage at the caspase-3 and caspase-6 cleavage sites. Currently we are testing whether skipping of this caspase-6 motif results in a reduction of N-terminal huntingtin fragments and thus reduced toxicity. Conclusions Above described preliminary results suggest a novel therapeutic approach to reduce toxic N-terminal huntingtin fragments using exon skipping while maintaining huntingtin protein levels. Currently we are testing in vivo whether a single bilateral injection of AONs in the mice striatum would result in skipping of the caspase motifs.