Using genetic variability in whole transcriptome expression in cells and tissues to understand the pathogenesis of Parkinson's and Alzheimer's disease

Lead Research Organisation: University College London
Department Name: Institute of Neurology

Abstract

Parkinson's and Alzheimer's disease (PD & AD) are common diseases that affect the brain, causing certain cells to die. When these cells are lost people experience, amongst other problems, difficulty remembering recent events in the case of AD and difficulties moving in the case of PD. At the moment these diseases affect over 1 million people in the United Kingdom with a devastating impact on patients and their families. Since both these diseases become more common with age, the number of sufferers is likely to increase as the number of elderly people rises in the UK. Unfortunately, at the moment we know relatively little about why certain people suffer from these diseases and until we do it is hard for researchers and doctors to develop effective treatments.
In the past 3 years scientists have been able to show that some individuals have common changes in their DNA (the instruction manual for all the building blocks needed to make the human body) that put them at higher risk of developing these conditions. Although this has been an amazing step forward, it has not been easy for scientists to understand how these changes in the DNA, termed genetic risk factors, act to help cause disease. Many people believe that one way in which these genetic risk factors might cause disease is by changing how much or the way in which particular genes (the basic building blocks) are expressed in brain cells. In order to check this, researchers need to make measurements about the genetic variation an individual carries and link this information to the genes they express in their cells.
Although this type of investigation is particularly challenging for diseases that affect the brain, it is possible and meeting those challenges is the aim of this project. One of the difficulties is accurately measuring the amount of the gene expressed in the right cells in the brain. The brain is extremely complex and even within a single region there are many different types of cells. We will address this problem by making use of a technique called laser capture microdissection to obtain pure collections of cells from brain regions relevant to PD and AD. In this technique, using very thin slices of the human brain tissue, we shoot out just the cells we are interested in collecting with a high power laser and then extract the RNA (the gene copies) from the pellets we shoot out. Another advance is the advent of new technology for measuring all the genes expressed in a single cell type or tissue sample. This technology is called "RNA-seq" or "whole transcriptome sequencing" and it allows us to measure all kinds of gene product from any gene. It also allows us to measure the relative quantities of alternative versions of the same gene, and to measure these quantities in such a way that the influence of genetic risk factors can be more sensitively detected by directly comparing the relative product quantities in individuals who happen to have both a "good" and a "bad" copy of a given genetic variant.
We believe that we are in an ideal position to complete this project and so add important insights to the understanding of PD and AD. We already have many of the RNA samples needed to perform this study and while some new RNA samples will be created during the project, through our close collaboration with the MRC Sudden Death Brain and Tissue Bank in Edinburgh we have the brain samples needed to do this (all of which have been donated for research). We have also already carried out extensive analysis of DNA from these samples, which means that the new information we generate during this grant can be quickly and cost-effectively added to existing sources of data in order to progress this research. As a final benefit, all the data we will generate on the human brain will be made publically available so that other scientists interested in understanding how the human brain works in health and disease can use this information.

Technical Summary

The aim of this study is to determine the effect of genetic risk factors on whole transcriptome expression in brain regions and cell types most vulnerable to Parkinson's and Alzheimer's disease (PD and AD) in order to understand the pathogenic processes underlying these conditions. This project is inspired by the recent discovery of 15 new genetic risk variants for PD and 13 new loci for AD. However, knowing the genomic position of risk variants is not equivalent to knowing how they act in terms of the underlying molecular processes or where they act in terms of the regional or cellular location. We aim to address both these issues. Using control post-mortem human brain tissue originating from 150 individuals, we will use expression quantitative trait locus (eQTL) mapping and allele-specific expression (ASE) analysis to identify loci that both change disease risk and regulate the expression of specific gene transcripts within specific tissues/cells. We will conduct these studies within the tissues and cells most relevant to PD and AD, namely the substantia nigra, hippocampus, temporal cortex, dopaminergic neurons, CA1 neurons, pyramidal neurons and astrocytes. Existing exome sequencing and SNP array data will be combined with whole transcriptome RNA sequencing data generated on the Illumina HiSeq2000 platform for downstream analysis. These two types of information - genetic variation and transcript expression - will be used for eQTL mapping in tissues and ASE analysis in cells. In both cases the eQTLs identified will be annotated for disease-relevance in the first instance. All the data generated by this project will be publicly released as both raw data files for re-analysis and processed information suitable for non-expert users through NCBI's Gene Expression Omnibus repository and Phenotype-Genotype Integrator sites. Thus, we will generate novel disease-relevant findings and provide the neuroscience community with a world-class resource.

Planned Impact

The goal of this work is to develop comprehensive databases which allow the interpretation of the genome wide association studies (GWAS) for the two most common neurodegenerative diseases, Parkinson's and Alzheimer's disease (PD and AD). In this way, we will improve our understanding of the pathogenic processes underlying these diseases and in the long term improve the diagnosis and treatment of patients struggling to cope with these debilitating diseases. Reaching this goal will require many intermediate steps and we describe who we envisage using our research findings and how this data will help them.

Researchers interested in the follow up of GWAS data: This study was designed to improve our understanding of the molecular mode of action and cellular location in which genetic risk variants for PD and AD act. Therefore, the most obvious users of our data are the original AD and PD consortia. However, the results of this study will also be useful for the interpretation of GWAS for other neurological and psychiatric disorders, making it relevant to researchers interested in the results of GWAS in frontotemporal dementia, epilepsy, depression and schizophrenia to name a few. Since we are publicly releasing all findings through the NCBI's searchable database, Phenotype-Genotype Integrator, we envisage other less specialist researchers also accessing and using the data. These individuals include clinicians, epidemiologists and drug companies interested in following up GWAS hits for clinical trial design or biomarker development.

Researchers interested in the creation and use of in vitro model systems: The cell-specific whole transcriptome sequencing data produced in this project will be a baseline for the detailed molecular assessment of stem cell-based disease models. We will work collaboratively with researchers in the University of Edinburgh (Dr. Kunath & Dr. Chandran) and Cambridge University (Dr. Livesey) to identify the strengths and weaknesses of existing cell models and work to improve the accuracy of these systems. Thus, we will contribute to the development of new research tools for use by the neuroscience research community within universities and in the private setting.

Basic neurobiologists: The large gene expression data sets we are producing will provide insights into other aspects of basic neurobiology. For example, it will be of value to those researchers interested in analysing sex differences in transcript expression. Using this data it will be possible to improve our understanding of the basis of well recognised differences in the incidence, presentation and response to treatment of many neurological disorders. This could in turn contribute to the drive towards individualised patient assessment and care.

Bioinformaticians & statistical geneticists: The raw genotyping, exome and RNA sequencing data will be used by statistical genetics and bioinformatics wishing to test and develop new statistical and analytical methods. In particular, the existence of paired exome sequencing and SNP array data will allow researchers to objectively test and improve methods for the imputation of genetic data. Therefore, we will directly contribute to one of the MRC's stated aims, the training of more researchers and clinicians in bioinformatics.

Charities & patient groups: The discovery of genetic risk variants for PD and AD represent one of the most significant steps forward in the understanding of these diseases in the last 10 years. Therefore, they are hugely important to patients and carers who desperately want to see progress in the diagnosis and treatment of their diseases. From our own experience of hosting visits for Parkinson's Disease UK, Alzheimer's Research UK and the Michael J Fox Foundation, we recognise the importance of engaging with patients and communicating the findings generated from our research in order to maintain hope, create novel resources and improve the uptake of clinical trials.

Publications

10 25 50
 
Description Attachment with Genomics England
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description House of Lords visit
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
Impact Lobbying for more funding for movement disorder diseases
 
Description Invited to attend a workshop on skills development for the NIHR
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description Joint Lead for Bioinformatics in Neurology GeCIP
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description EU Joint Programme Grant
Amount € 530,000 (EUR)
Organisation JPND Research 
Sector Academic/University
Country Global
Start 01/2014 
End 01/2017
 
Description Project Grant
Amount £1,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 03/2010 
End 03/2013
 
Description Project Grant
Amount £1,300,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 03/2013 
End 03/2016
 
Description Wellcome Biomedical Resource Grant
Amount £200,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2016 
End 10/2019
 
Title A web-based tool for quality control of eQTL data 
Description This is a web-based tool to allow users to check whether reported eQTLs may be due to systematic errors arising from the presence of polymorphisms within expression microarray probes. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact This is currently being used by our collaborators based in the NIH, but will soon be made publicly available. It has resulted in the identification of a significant number of false positive results. 
 
Title A web-based tool for eQTL analysis and expression profiles 
Description We have created a web-based tool to allow researchers to access the eQTL data we have generated and expression profiles in human brain. 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact This is presently being used by groups within KCL and UCL to follow up GWAS results. 
 
Title Database on genetic variation in neurologically normal individuals over the age of 60 years old 
Description I contributed to the HEXdatabase which provides information on genetic variation in individuals with no evidence of a neurological disorder. 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact This tool will be released on the Alzform website. 
 
Title Genome-wide gene expression in 10 human brain regions 
Description This is a database of exon-specific gene expression generated from over 800 RNA samples extracted from post-mortem human brain tissue. 
Type Of Material Biological samples 
Provided To Others? No  
Impact This collection of data when released will provide researchers working in the field of neuroscience comprehensive imformation on the expression of all genes within multiple human brain regions. 
 
Title Human genome expression database 
Description Gene expression database. http://caprica.genetics.kcl.ac.uk:51519/BRAINEAC/ 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2010 
Provided To Others? Yes  
Impact It has been accessed several thousand times and data from it incorporated into at least 100 publications 
URL http://smarturl.it/braineac
 
Title StemBANCC 
Description Stem cell biobank. Freely available. 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact This has been a successful application and many drug companies are using stem cell lines supplied by our group 
URL http://stembancc.org
 
Description Age-related changes in gene expression in human brain 
Organisation Wellcome Trust
Department Multiple Tissue Human Expression Resource (MuTHER) consortium
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution We have provided gene expression data on human brain.
Collaborator Contribution They have provided gene expression data relating to multiple other human tissues.
Impact There is a paper in submission with Genome Research
Start Year 2011
 
Description Collection and analysis of control human post-mortem brain tissue 
Organisation University of Edinburgh
Department Neuropathology Unit
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have supplied our collaborators with information regarding the degree of RNA preservation within banked control human brain tissue.
Collaborator Contribution This collaboration has allowed to collect high quality control human brain tissue for analysis.
Impact This collaboration has allowed us to build a database of exon-specific gene expression in multiple human brain regions.
Start Year 2009
 
Description Genetic risk variants for mesio-temporal lobe epilepsy 
Organisation University College London
Department Institute of Neurology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have provided regional eQTL data and total RNA extracted from human brain tissue originating from neurologically normal individuals.
Collaborator Contribution They have identified risk SNPs for mesio-temporal lobe epilepsy.
Impact There is presently a paper in submission with Nature Genetics.
Start Year 2011
 
Description PARK7 gene structure and expression 
Organisation University of Liverpool
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Expression and RNAseq data and data interpretation relating to PARK7 expression in human brain
Collaborator Contribution Expertise regarding the structure and function of PARK7
Impact Manuscript in preparation
Start Year 2012
 
Description Whole genome expression analysis for the assessment of hESC-derived neuronal maturation 
Organisation University of Edinburgh
Department Centre for Clinical Brain Sciences (CCBS)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution I have performed the expression and splicing analysis of these samples.
Collaborator Contribution They have provided samples of hESCs, neural precursors and hESc-derived neurons. They have also provided expertise on the process of neural differentiation.
Impact We currently have a paper in submission to Human Molecular Genetics. This collaboration is multidisciplinary with specialist cell and developmental biologists.
Start Year 2011
 
Description Whole genome expression database 
Organisation National Institute on Aging
Department Laboratory of Neurogenetics
Country United States of America 
Sector Public 
PI Contribution We are just abouhttp://bit.ly/braineac (ignore certificate warning) username: UKBEC password: BraineacNOW t to relase this database to the general public
Collaborator Contribution We worked with our Us collaborators on this project (Singleton/Cookson): about half the work each
Impact Just search Ryten plus Singleton on pubmed These are the current PMID 23223016, 23177596, 22892372 , 22777693, 22723018, 22433082, 21738488 , 21292315
Start Year 2009
 
Description Whole genome expression in iPS-derived neurons in health and disease 
Organisation University of Edinburgh
Department MRC Centre for Regenerative Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution We have performed the analysis of geen expression and splicing in these samples.
Collaborator Contribution It has resulted in the provision of unique samples for the investigation of the impact of specific genetic mutations known to cause Parkinson's Disease on gene expression.
Impact This collaboration has resulted in a publication in Nature Communications and also prompted the submission of a successful project grant to the Alzheimer's Research Trust (£30,000). This is a project using the skills and knowledge of specialist cell biologists with my own technical skills in expression analysis.
Start Year 2011
 
Description eQTL analysis in control human cerebellum and frontal cortex 
Organisation National Center for Biotechnology Information (NCBI)
Country United States of America 
Sector Public 
PI Contribution We have provided a considerable amount of processed gene expression data.
Collaborator Contribution This collaboration has allowed us to increase the power of our study by increasing the n number and has provided us with technical expertise regarding the analysis of eQTL data.
Impact We have a publication in submission as a direct result of this study.
Start Year 2010
 
Description eQTLs with relevance to brain anatomy as measured by MRI 
Organisation National Center for Biotechnology Information (NCBI)
Country United States of America 
Sector Public 
PI Contribution We have provided region-specific eQTL data.
Collaborator Contribution The ENIGMA consortium have used MRI scanning and SNP genotyping to identify SNPs that relate to hippocampal volume among other measurements.
Impact This collaboration has resulted in the publication of a Nature Genetics paper.
Start Year 2011
 
Description Talk for the Cardiff Gene Park to sixthformers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact The talk sparked discussion following the event

No notable impact yet
Year(s) Of Engagement Activity 2014
 
Description Website - Alzforum 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview has sparked online comments

Requests for data sharing
Year(s) Of Engagement Activity 2014
 
Description Website - SAFARI 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The report has sparked interest in our data set and resources

I have been e-mailed for specific data
Year(s) Of Engagement Activity 2014
 
Description Workshop on Alzheimer's Disease and Related Disorders, FBRI Meeting, Kronberg, Germany 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation workshop facilitator
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Prof Hardy led the workshop. Exchange of ideas and information.

Excellent feedback. Participants reported being pleased with the intellectual stimulation.
Year(s) Of Engagement Activity 2015