[Show abstract][Hide abstract] ABSTRACT: Coeliac disease (CeD) is a highly heritable common autoimmune disease involving chronic small intestinal inflammation in response to dietary wheat. The human leukocyte antigen (HLA) region, and 40 newer regions identified by genome wide association studies (GWAS) and dense fine mapping, account for ∼40% of the disease heritability. We hypothesized that in pedigrees with multiple individuals with CeD rare [minor allele frequency (MAF) <0.5%] mutations of larger effect size (odds ratios of ∼ 2-5) might exist. We sequenced the exomes of 75 coeliac individuals of European ancestry from 55 multiply affected families. We selected interesting variants and genes for further follow up using a combination of: an assessment of shared variants between related subjects, a model-free linkage test, and gene burden tests for multiple, potentially causal, variants. We next performed highly multiplexed amplicon resequencing of all RefSeq exons from 24 candidate genes selected on the basis of the exome sequencing data in 2,248 unrelated coeliac cases and 2,230 controls. 1,335 variants with a 99.9% genotyping call rate were observed in 4,478 samples, of which 939 were present in coding regions of 24 genes (Ti/Tv 2.99). 91.7% of coding variants were rare (MAF <0.5%) and 60% were novel. Gene burden tests performed on rare functional variants identified no significant associations (p<1×10-3) in the resequenced candidate genes. Our strategy of sequencing multiply affected families with deep follow up of candidate genes has not identified any new CeD risk mutations.
[Show abstract][Hide abstract] ABSTRACT: There are two major pathways leading to induction of NF-kB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IkBa in a manner dependent on IkB kinase (IKK) b and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-kB2(p52)/RelB containing complexes, and is dependent on IKKa and NF-kB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-kB pathway subunits NF-kB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-kB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-kB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-kB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-kB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-kB can inhibit tumor suppressor function and promote tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrPC). They propagate by recruiting host-encoded PrPC although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrPC expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrPC deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state.
[Show abstract][Hide abstract] ABSTRACT: Prion diseases (transmissible spongiform encephalopathies) are fatal neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and bovine spongiform encephalopathy (BSE) in cattle. While genome-wide association studies in human and quantitative trait loci mapping in mice have provided evidence for multiple susceptibility genes, few of these have been confirmed functionally. Phenotyping mouse models is generally the method of choice. However, this is not a feasible option where many novel genes, without pre-existing models, would need to be tested. We have therefore developed and applied an in-vitro screen to triage and prioritise candidate modifier genes for more detailed future studies which is faster, far more cost effective and ethical relative to mouse bioassay models. An in vitro prion bioassay, the scrapie cell assay (SCA), uses a neuroblastoma derived cell line (PK1) that is susceptible to RML prions and able to propagate prions at high levels. In this study, we have generated stable gene silencing and/or overexpressing PK1-derived cell lines to test whether perturbation of 14 candidate genes affects prion susceptibility. While no consistent differences were determined for seven genes, highly significant changes were detected for Zbtb38, Sorcs1, Stmn2, Hspa13, Fkbp9, Actr10 and Plg, suggesting that they play key roles in the fundamental processes of prion propagation or clearance. Many neurodegenerative diseases involve the accumulation of misfolded protein aggregates and "prion-like" seeding and spread has been implicated in their pathogenesis. It is therefore expected that some of these prion-modifier genes may be of wider relevance in neurodegeneration.
Full-text · Article · May 2014 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.
[Show abstract][Hide abstract] ABSTRACT: Prion infections, causing neurodegenerative conditions such as Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep and BSE in cattle are characterised by prolonged and variable incubation periods that are faithfully reproduced in mouse models. Incubation time is partly determined by genetic factors including polymorphisms in the prion protein gene. Quantitative trait loci studies in mice and human genome-wide association studies have confirmed that multiple genes are involved. Candidate gene approaches have also been used and identified App, Il1-r1 and Sod1 as affecting incubation times. In this study we looked for an association between App, Il1-r1 and Sod1 representative SNPs and prion disease incubation time in the Northport heterogeneous stock of mice inoculated with the Chandler/RML prion strain. No association was seen with App, however, significant associations were seen with Il1-r1 (P = 0.02) and Sod1 (P<0.0001) suggesting that polymorphisms at these loci contribute to the natural variation observed in incubation time. Furthermore, following challenge with Chandler/RML, ME7 and MRC2 prion strains, Sod1 deficient mice showed highly significant reductions in incubation time of 20, 13 and 24%, respectively. No differences were detected in Sod1 expression or activity. Our data confirm the protective role of endogenous Sod1 in prion disease.
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are fatal neurodegenerative disorders that include bovine spongiform encephalopathy (BSE) and scrapie in animals and Creutzfeldt-Jakob disease (CJD) in humans. They are characterized by long incubation periods, variation in which is determined by many factors including genetic background. In some cases it is possible that incubation time may be directly correlated to the level of gene expression. To test this hypothesis, we combined incubation time data from five different inbred lines of mice with quantitative gene expression profiling in normal brains and identified five genes with expression levels that correlate with incubation time. One of these genes, Hspa13 (Stch), is a member of the Hsp70 family of ATPase heat shock proteins, which have been previously implicated in prion propagation. To test whether Hspa13 plays a causal role in determining the incubation period, we tested two overexpressing mouse models. The Tc1 human chromosome 21 (Hsa21) transchromosomic mouse model of Down syndrome is trisomic for many Hsa21 genes including Hspa13 and following Chandler/Rocky Mountain Laboratory (RML) prion inoculation, shows a 4% reduction in incubation time. Furthermore, a transgenic model with eightfold overexpression of mouse Hspa13 exhibited highly significant reductions in incubation time of 16, 15, and 7% following infection with Chandler/RML, ME7, and MRC2 prion strains, respectively. These data further implicate Hsp70-like molecular chaperones in protein misfolding disorders such as prion disease.
Full-text · Article · Aug 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Genetic heterogeneity is common in many neurologic disorders. This is particularly true for the hereditary ataxias where at least 36 disease genes or loci have been described for spinocerebellar ataxia and over 100 genes for neurologic disorders that present primarily with ataxia. Traditional genetic testing of a large number of candidate genes delays diagnosis and is expensive. In contrast, recently developed genomic techniques, such as exome sequencing that targets only the coding portion of the genome, offer an alternative strategy to rapidly sequence all genes in a comprehensive manner. Here we describe the use of exome sequencing to investigate a large, 5-generational British kindred with an autosomal dominant, progressive cerebellar ataxia in which conventional genetic testing had not revealed a causal etiology.
Twenty family members were seen and examined; 2 affected individuals were clinically investigated in detail without a genetic or acquired cause being identified. Exome sequencing was performed in one patient where coverage was comprehensive across the known ataxia genes, excluding the known repeat loci which should be examined using conventional analysis.
A novel p.Arg26Gly change in the PRKCG gene, mutated in SCA14, was identified. This variant was confirmed using Sanger sequencing and showed segregation with disease in the entire family.
This work demonstrates the utility of exome sequencing to rapidly screen heterogeneous genetic disorders such as the ataxias. Exome sequencing is more comprehensive, faster, and significantly cheaper than conventional Sanger sequencing, and thus represents a superior diagnostic screening tool in clinical practice.
[Show abstract][Hide abstract] ABSTRACT: In-vitro endpoint titration of RML 6200 using SCEPA. Serially diluted RML 6200 was transferred onto layers of prion-susceptible PK1 cells and the number of positive and negative wells was determined by SCEPA as described in Materials and Methods. The complementary log-log transformed data were plotted in Figure S1.
[Show abstract][Hide abstract] ABSTRACT: Infectious titers of MACS-isolated splenic cell types in the absence of prion replication at 3 dpi. A group of four Prnp−/− mice were inoculated intraperitoneally with 100 µl 1% (w/v) RML I6200 and spleens were dissected at 3 dpi. Different cell types were isolated by magnetic sorting, infectious titers determined by SCEPA and titers estimated by GLM.
[Show abstract][Hide abstract] ABSTRACT: Immunohistological characterisation of scrapie-infected spleen tissue at early stages after infection. An increase in the number of lymphoid follicles containing follicular dendritic cells containing abnormal prion protein (ICSM35 immunostaining) as well as an increase in the density of PrPSc deposition is seen with increasing incubation time. Positive follicles were determined as the ratio of the number of ICSM35-positive follicles and the total number of follicles (counted on an adjacent H&E section. PrPSc density in follicles was determined semi-quantitatively as weak (shown in Figure 3C, 3 and 7 dpi), moderate (Figure 3C, 14 and 30 dpi) and strong.
[Show abstract][Hide abstract] ABSTRACT: DCs are not activated at preclinical stages of Scrapie. 129Sv×C57BL/6 mice were inoculated i.p. with 100 µl 1% RML I6200 or 100 µl 1% uninfected CD1 homogenate and splenocytes were isolated at 110 dpi. DCs were isolated by MACS using a 1∶1 mix of CD11 and mPDCA-1 microbeads and labelled with a specific mAb against CD86. No evidence for an expression difference of CD86 between scrapie-infected and age-matched control mice were detected at preclinical stages.
[Show abstract][Hide abstract] ABSTRACT: No abnormalities of splenic B cell subsets at preclinical disease. 29Sv×C57BL/6 mice were inoculated i.p. with 100 µl 1% RML I6200 or 100 µl 1% uninfected CD1 homogenate and culled at 80 dpi (A) and 100 dpi (B). Splenocytes were isolated according to Materials and Methods. To analyse B cell subsets splenocytes were labelled with mAbs against anti-CD19, anti-CD23 and anti-CD21. The CD19-gated B cell population was examined for CD21/35 and CD23 expression. No alterations were detected in the ratios of CD21high D23- marginal zone (MZ) and CD21int D23high follicular (FO) B cells.
[Show abstract][Hide abstract] ABSTRACT: Initial validation of Poisson distribution. To check whether the experimental data from in-vitro endpoint titrations indicate an underlying Poisson distribution for the number of infected cells serial 1∶3 dilutions of RML brain homogenate within a range between 10-7 and 10-9 were prepared and cell layers of 12 wells per dilution were infected. Complementary log-log transformed proportions of negative wells are shown for eight technical assay repeats. Linear regression analysis was performed for dilutions were the proportion of positive wells for all eight repeats were >0 and <12 per total number of wells and a slope factor β of 1.06±0.20 was calculated.
[Show abstract][Hide abstract] ABSTRACT: Protein expression levels of PrPc are undetectable in pDCs of 129Sv×C57BL/6 mice. PDCs isolated from uninfected 129Sv×C57BL/6 or Prnp−/− mice were labeled with biotinylated monoclonal anti-PrP antibody ICSM 35 followed by allophycocyanin (APC)-streptavidin and PrPc expression levels were analysed by flow cytometry. No difference in PrPc expression levels between pDCs from 129Sv×C57BL/6 (wildtype) and Prnp−/− mice was detected. As a control for PrPc expression mouse neuroblastoma cells (N2a) were labeled with biotinylated ICSM35 and biotinylated mouse IgG2b isotype control.