Holger Hummerich

Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia.

We have previously reported a large Danish pedigree with autosomal dominant frontotemporal dementia (FTD) linked to chromosome 3 (FTD3). Here we identify a mutation in CHMP2B, encoding a component of the endosomal ESCRTIII complex, and show that it results in aberrant mRNA splicing in tissue samples from affected members of this family. We also describe an additional missense mutation in an unrelated individual with FTD. Aberration in the endosomal ESCRTIII complex may result in FTD and neurodegenerative disease.

Genetic analysis of the cytoplasmic dynein subunit families.

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study

Summary Background Human and animal prion diseases are under genetic control, but apart from PRNP (the gene that encodes the prion protein), we understand little about human susceptibility to bovine spongiform encephalopathy (BSE) prions, the causal agent of variant Creutzfeldt–Jakob disease (vCJD). Methods We did a genome-wide association study of the risk of vCJD and tested for replication of our findings in samples from many categories of human prion disease (929 samples) and control samples from the UK and Papua New Guinea (4254 samples), including controls in the UK who were genotyped by the Wellcome Trust Case Control Consortium. We also did follow-up analyses of the genetic control of the clinical phenotype of prion disease and analysed candidate gene expression in a mouse cellular model of prion infection. Findings The PRNP locus was strongly associated with risk across several markers and all categories of prion disease (best single SNP [single nucleotide polymorphism] association in vCJD p=2·5×10−17; best haplotypic association in vCJD p=1×10−24). Although the main contribution to disease risk was conferred by PRNP polymorphic codon 129, another nearby SNP conferred increased risk of vCJD. In addition to PRNP, one technically validated SNP association upstream of RARB (the gene that encodes retinoic acid receptor beta) had nominal genome-wide significance (p=1·9×10−7). A similar association was found in a small sample of patients with iatrogenic CJD (p=0·030) but not in patients with sporadic CJD (sCJD) or kuru. In cultured cells, retinoic acid regulates the expression of the prion protein. We found an association with acquired prion disease, including vCJD (p=5·6×10−5), kuru incubation time (p=0·017), and resistance to kuru (p=2·5×10−4), in a region upstream of STMN2 (the gene that encodes SCG10). The risk genotype was not associated with sCJD but conferred an earlier age of onset. Furthermore, expression of Stmn2 was reduced 30-fold post-infection in a mouse cellular model of prion disease. Interpretation The polymorphic codon 129 of PRNP was the main genetic risk factor for vCJD; however, additional candidate loci have been identified, which justifies functional analyses of these biological pathways in prion disease. Funding The UK Medical Research Council.

A Novel Protective Prion Protein Variant that Colocalizes with Kuru Exposure

BACKGROUND Kuru is a devastating epidemic prion disease that affected a highly restricted geographic area of the Papua New Guinea highlands; at its peak, it predominantly affected adult women and children of both sexes. Its incidence has steadily declined since the cessation of its route of transmission, endocannibalism. METHODS We performed genetic and selected clinical and genealogic assessments of more than 3000 persons from Eastern Highland populations, including 709 who participated in cannibalistic mortuary feasts, 152 of whom subsequently died of kuru. RESULTS Persons who were exposed to kuru and survived the epidemic in Papua New Guinea are predominantly heterozygotes at the known resistance factor at codon 129 of the prion protein gene (PRNP). We now report a novel PRNP variant--G127V--that was found exclusively in people who lived in the region in which kuru was prevalent and that was present in half of the otherwise susceptible women from the region of highest exposure who were homozygous for methionine at PRNP codon 129. Although this allele is common in the area with the highest incidence of kuru, it is not found in patients with kuru and in unexposed population groups worldwide. Genealogic analysis reveals a significantly lower incidence of kuru in pedigrees that harbor the protective allele than in geographically matched control families. CONCLUSIONS The 127V polymorphism is an acquired prion disease resistance factor selected during the kuru epidemic, rather than a pathogenic mutation that could have triggered the kuru epidemic. Variants at codons 127 and 129 of PRNP demonstrate the population genetic response to an epidemic of prion disease and represent a powerful episode of recent selection in humans.

Activation of Nuclear Factor-kappa B signalling promotes cellular senescence

Cellular senescence is a programme of irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress. The underlying signalling pathways, of major clinicopathological relevance, are unknown. We combined genome-wide expression profiling with genetic complementation to identify genes that are differentially expressed when conditionally immortalised human fibroblasts undergo senescence upon activation of the p16-pRB and p53-p21 tumour suppressor pathways. This identified 816 up and 961 downregulated genes whose expression was reversed when senescence was bypassed. Overlay of this data set with the meta-signatures of genes upregulated in cancer showed that nearly 50% of them were downregulated upon senescence showing that even though overcoming senescence may only be one of the events required for malignant transformation, nearly half of the genes upregulated in cancer are related to it. Moreover 65 of the up and 26 of the downregulated genes are known downstream targets of nuclear factor (NF)-κB suggesting that senescence was associated with activation of the NF-κB pathway. Direct perturbation of this pathway bypasses growth arrest indicating that activation of NF-κB signalling has a causal role in promoting senescence.

HTT-lowering reverses Huntington’s disease immune dysfunction caused by NFκB pathway dysregulation

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntingtons 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 Huntingtons 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 Huntingtons 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 Huntingtons disease.

HECTD2 Is Associated with Susceptibility to Mouse and Human Prion Disease

Prion diseases are fatal transmissible neurodegenerative disorders, which include Scrapie, Bovine Spongiform Encephalopathy (BSE), Creutzfeldt-Jakob Disease (CJD), and kuru. They are characterised by a prolonged clinically silent incubation period, variation in which is determined by many factors, including genetic background. We have used a heterogeneous stock of mice to identify Hectd2, an E3 ubiquitin ligase, as a quantitative trait gene for prion disease incubation time in mice. Further, we report an association between HECTD2 haplotypes and susceptibility to the acquired human prion diseases, vCJD and kuru. We report a genotype-associated differential expression of Hectd2 mRNA in mouse brains and human lymphocytes and a significant up-regulation of transcript in mice at the terminal stage of prion disease. Although the substrate of HECTD2 is unknown, these data highlight the importance of proteosome-directed protein degradation in neurodegeneration. This is the first demonstration of a mouse quantitative trait gene that also influences susceptibility to human prion diseases. Characterisation of such genes is key to understanding human risk and the molecular basis of incubation periods.

Genome-wide association study in multiple human prion diseases suggests genetic risk factors additional to PRNP

Prion diseases are fatal neurodegenerative diseases of humans and animals caused by the misfolding and aggregation of prion protein (PrP). Mammalian prion diseases are under strong genetic control but few risk factors are known aside from the PrP gene locus (PRNP). No genome-wide association study (GWAS) has been done aside from a small sample of variant Creutzfeldt-Jakob disease (CJD). We conducted GWAS of sporadic CJD (sCJD), variant CJD (vCJD), iatrogenic CJD, inherited prion disease, kuru and resistance to kuru despite attendance at mortuary feasts. After quality control, we analysed 2000 samples and 6015 control individuals (provided by the Wellcome Trust Case Control Consortium and KORA-gen) for 491032-511862 SNPs in the European study. Association studies were done in each geographical and aetiological group followed by several combined analyses. The PRNP locus was highly associated with risk in all geographical and aetiological groups. This association was driven by the known coding variation at rs1799990 (PRNP codon 129). No non-PRNP loci achieved genome-wide significance in the meta-analysis of all human prion disease. SNPs at the ZBTB38-RASA2 locus were associated with CJD in the UK (rs295301, P = 3.13 × 10(-8); OR, 0.70) but these SNPs showed no replication evidence of association in German sCJD or in Papua New Guinea-based tests. A SNP in the CHN2 gene was associated with vCJD [P = 1.5 × 10(-7); odds ratio (OR), 2.36], but not in UK sCJD (P = 0.049; OR, 1.24), in German sCJD or in PNG groups. In the overall meta-analysis of CJD, 14 SNPs were associated (P < 10(-5); two at PRNP, three at ZBTB38-RASA2, nine at nine other independent non-PRNP loci), more than would be expected by chance. None of the loci recently identified as genome-wide significant in studies of other neurodegenerative diseases showed any clear evidence of association in prion diseases. Concerning common genetic variation, it is likely that the PRNP locus contains the only strong risk factors that act universally across human prion diseases. Our data are most consistent with several other risk loci of modest overall effects which will require further genetic association studies to provide definitive evidence.

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Exome sequencing in an SCA14 family demonstrates its utility in diagnosing heterogeneous diseases

Objective: 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. Methods: 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. Results: 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. Conclusions: 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.