Prediction and analysis of a regulatory SNP map of Major Depressive Disorder

Lead Research Organisation: University of Aberdeen
Department Name: Cal Unit (Medical Faculty)

Abstract

Major depressive disorder (MDD) constitutes the most frequently occurring mental health problem in the world and will affect up to 25% of us during our lifetimes. Worryingly, MDD is on the increase.
This study will set out to find the genetic causes of MDD by examining changes in the DNA sequence of people who are more susceptible to depression. In addition, this study will determine why certain MDD sufferers do not respond to current treatments.
Previous studies of this type have examined possible changes in the regions the genome that make protein.
Recently, however, it has been demonstrated that the majority of differences between individuals, including disease susceptibility, may be caused by changes, not in the genes themselves, but in the poorly understood DNA sequences that act as switches for these genes. These switches ensure that genes essential to healthy brain function are only used in the correct parts of the brain at the proper times and in the right dose.
Unlike genes, little is known of these switches as, up to now they have been very hard to find. However, teams led by Dr. MacKenzie have had considerable success in identifying switches responsible for driving the expression of genes known to be involved in depression, addiction and inflammatory pain.
This project will combine Dr. Mackenzies ability to detect important switch sequences, that make up less than 5% of the genome, with our new ability to detect harmful mutations within these switches. Professor McGuffin and Dr. Breen will then analyse the DNA of over 1000 individuals suffering MDD to determine whether the occurrence of particular switch differences can be associated with susceptibility to MDD. Dr MacKenzie and Prof. Quinn will then carry out molecular biological studies of these switch differences to determine the biochemical pathways affected. In addition, Prof Quinn will evaluate how the activity of variants of these switches are affected by a number of known antidepressant drugs to determine the genetic causes of variation in MDD drug treatments.
By combining a number of newly developed computer based, patent sample based and molecular biology techniques this study has the real potential of hugely expanding our understanding of the mechanisms regulating the use of key genes in the brain and how changes on these mechanisms may contribute to susceptibility to MDD. Furthermore, this study will allow us to examine why many MDD sufferers do not respond to current anti-depressive medications.

Technical Summary

Major Depressive Disorders (MDD) make up the most prevalent class of psychiatric disorders and will affect ~25% of us. The social and economic costs of these disorders are enormous with an estimated loss to the UK economy alone of some #9 billion per year. An additional problem facing the treatment of sufferers is variability in the efficacy of the drugs used to treat MDD.
These disorders have a strong genetic basis. Although many of these studies are still ongoing it is becoming clear that studying the effects of polymorphisms on coding regions alone will not give us a complete picture of the underlying causes.
We will explore the hypothesis that susceptibility to MDD and variation in drug efficacy may also involve polymorphisms within the gene regulatory regions responsible for maintaining normal gene expression of components of systems including the serotoninergic, dopaminergic, tachykininergic, and NPY pathways.
The objectives of this project will be to use a predictive systems biology approach to identify regulatory regions within the human genome by comparative genomics. The tissue specific and inducible status of these enhancer candidates will be analysed initially in primary cell lines and subsequently in transgenic animals in both the MacKenzie and Quinn labs.
By concentrating on SNPs within highly conserved and thus functional regions of the genome this novel approach will greatly accelerate the association of SNPs within these regions with susceptibility to MDD that will be examined in large MDD patient samples in the Institute of Psychiatry by a team headed by McGuffin and Breen.
Molecular analysis of protein-DNA interactions affected by these polymorphic ECRs will also be examined using a combination of EMSA and ChIP assay in the MacKenzie and Quinn labs. Finally, translational pharmacogenomic studies of the affects of a number of different psychoactive compounds on positively associated regulatory polymorphisms will be further examined using primary culture and in transgenic animals in the Quinn lab.
This is a novel, ambitious and multi-disciplined approach involving a unique collaboration between 3 laboratories that are individually recognised as being international leaders in their respective fields. By applying a unique combination of systems biology, patient based association study and translational pharmacogenomics this exciting collaboration has the potential of changing our understanding of the causes of MDD and will act as a translational platform that will allow for the development of more effective and specific anti-depressive therapies.

Publications

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Ball HA (2009) Genetic and environmental contributions to depression in Sri Lanka. in The British journal of psychiatry : the journal of mental science

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Allan CL (2009) Twin study of illness history variables in psychosis. in Schizophrenia research

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Ball HA (2009) Depression, migraine with aura and migraine without aura: their familiality and interrelatedness. in Cephalalgia : an international journal of headache

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Perroud N (2009) Genetic predictors of increase in suicidal ideation during antidepressant treatment in the GENDEP project. in Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

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Samaan Z (2009) Migraine in recurrent depression: case-control study. in The British journal of psychiatry : the journal of mental science

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Uher R (2009) Differential efficacy of escitalopram and nortriptyline on dimensional measures of depression. in The British journal of psychiatry : the journal of mental science

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Ali FR (2010) Assessing the impact of genetic variation on transcriptional regulation in vitro. in Methods in molecular biology (Clifton, N.J.)

 
Description A novel in-vivo CRISPR enhancer screen to identify polymorphic regulatory regions controlling the hypothalamic expression of appetite regulating neuropeptides.
Amount £25,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 04/2018
 
Description Determining the effects of genetic variation and early life stress on the regulation of the galanin gene in fat and alcohol selection.
Amount £418,914 (GBP)
Funding ID BB/N017544/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Effects of stress, genetic and epigenetic variation on the control of the cannaboinnoid receptor gene; possible roles in disease and drug response stratification.
Amount £104,000 (GBP)
Organisation Medical Research Scotland 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description Wellcome trust ISSF fund
Amount £20,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Pump Prime Award
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 03/2016
 
Title BDNF promoter 4 lacZ transgenic line. 
Description Several transgenic lines werw generated using a plasmid containing the BDNF promoter 4 fused to the LacZ gene. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Non-coding polymorphisms within the BDNF gene have been associated with conditions as diverse as MDD, obesity and BD. In addition removal of BDNF promoter 4 has been shown to cause a reduction in cognition and memory in mice. However, Nothing is known of how many of the non-coding SNPs in and around the BDNF locus modulates the regulation of the BDNF gene. Using comparitive genomics we identified and isolated the BDNF gene promoter. This promoter was used to make reporter constructs which were then used to make transgenic mouse lines. These transgenic mouse lines expressed lacZ in many areas of the limbic system including the amygdala, hippocampus and cortex. By establishing the BDNF promoter-4 as being tissue specific we have established a platform for establishing the possible effects of non-coding SNPs in and around the BDNF gene on the activity of its promoter in amygdala, hippocampal and cortical cells. 
 
Title BICC1 overexpressing cell line 
Description BicC1 over expression A plasmid expressing a myc/DDK-tagged human BicC1 protein driven by a CMV promoter is being transfected into human neuroblastoma and HEK293 clonal lines. The mRNA message and protein expressed from this construct is being assessed by QPCR and western blotting, respectively, to determine the functionality of the construct. Western blotting is being performed with anti myc and anti DDK antibodies to confirm expression of the tagged protein. In addition, transfected cells are also being tested with the BicC1 antibodies to help boost the signal for optimising these antibodies. The tagged construct will allow determination of the localisation of the BicC1 protein within the cells. BicC1 has mostly been considered as an RNA binding protein and is chiefly located in the cytoplasm in other species. It is not yet been determined whether BicC1 can act as a DNA binding protein although it has KH domains similar to the hnRNPK single stranded nucleic acid binding protein which is a known regulator of DNA transcription. Localisation studies in combination with ChIP assays will go some way to determine whether BicC1 can act as a transcriptional regulator of gene expression in addition to its role in mRNA stability. Furthermore, changes in distribution of the BicC1 protein within the cellular environment will be assessed following specific challenges to the cells. 
Type Of Material Cell line 
Year Produced 2010 
Provided To Others? Yes  
Impact Having a reliable source of BICC1 protein produced from mammalian cells will be vital to efforts to characterise the function and mechamism of this protein in disease 
 
Title BICC1 promoter transgenic mice 
Description Transgenic lines containing the BICC1 promoter driving expression of the LacZ marker gene. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Non-coding polymorphisms within the BICC1 gene have been associated with MDD. Nothing is known of the regulation of the BICC1 gene. Using comparitive genomics we identified and isolated the BICC1 gene promoter. This promoter was used to make reporter constructs which were then used to make transgenic mouse lines. These transgenic mouse lines expressed lacZ in many areas of the limbic system including the amygdala, hippocampus and cortex. By establishing the BICC1 promoter as being tissue specific we have established a platform for establishing the possible effects of non-coding SNPs in and around the BICC1 gene on the activity of its promoter in amygdala, hippocampal and cortical cells. 
 
Title CACNA1c promoter transgenic line 
Description Transgenic lines produced by pro-nuclear microinjection 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact CACNA1c has been associated with bipolar disorder and schizophrenia by multiple GWAS analysis. By isolating and identifying the tissue specific properties of this promoter using transgenic analysis we have provided a platform for understanding the possible differential effects of non-coding SNPs on the expression of the CACNA1c gene in MDD. 
 
Title Expansion of the HPC array facility 
Description The MRC award allowed us to exapnd the capacity of our existing high performance computer (HPC) array. This has been of enormous benefit to our research effort and has profitted a number of other research groups around the University who require HPC array technology to carry out complex system biology type projects. 
Type Of Material Improvements to research infrastructure 
Year Produced 2008 
Provided To Others? Yes  
Impact The provision of an expanded HPC systems has resulted in the publication of a manuscript in the high profile journal BMC genomics. We also hope to publish a number of other papers in BMC bioinformatics in the near future. 
 
Title Magnetofection of primary neurones 
Description We optimized a novel method for the recovery, transfection and culture of primary neuonal cell lines derived from the amygdala, hypothalamus and hippocampus. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact The development of this technique removed much of the need to carry out experiments in whole animals. 
 
Title Rabbit anti human BICC1 antibody 
Description An antibody raised against the human BICC1 proteins 
Type Of Material Antibody 
Provided To Others? No  
Impact Detecting changes in the expression of the BICC1 protein will be critical to determining the mechanisms by which the BICC1 gene, that we have associated with major depressive disorder (MDD) using GWa studies, induces the symptoms of MDD. There are no commercial antibodies available against the human form Bicaudal C 1 homologue (BicC1). Two distinct antibodies raised in rabbit against the human homologue are being assessed for their potential to recognise the human form of BicC1 for future use in immunostaining and chromatin Immunoprecipitation assays. The human neuroblastoma cell lines used for assessing BicC1 ECR activity, SK-N-AS and SH SY5Y, were tested for the presence of BicC1 mRNA. A positive result in both cell lines suggested the presence of BicC1 in these cells lines. Furthermore, both the long isoform and the hypothetical short isoform were detected. Validation of the presence of BicC1 protein in these cell lines is now underway to confirm the existence of the BicC1 protein and to assess the antigenic potential of eight serum bleeds and four purified forms of the antibody that have been generated. Protein extracted from the neuroblastoma cell lines as well as that extracted from human embryonic kidney cells lines (HEK293), which should express BicC1 at high levels, is currently being used in western blot analysis. Initial results have shown a number of protein bands detected by both antibodies. One of these antibodies (recognising the epitope IKPKPKQPSKSVIVKSVER) would recognise both the long and the putative short isoform of BicC1. The purified from of this antibody gave a good signal with a band in the range of the expected size for the long form of BicC1. The second antibody that should only recognise the long isoform (recognising the epitope AAQGEPGYLAAQSDPGSNS), which gave poor ELISA results and was pooled from two sources, has failed to work in the purified form although serum bleeds of this antibody have identified a number of bands. Peptide competition of the antibody to determine specificity of the bands has thus far failed to compete bands although this is problematic for serum bleeds as antibody concentrations cannot be determined. For all antibodies generating bands one band in the kDa range for BicC1 appears consistently and this band is being prepared for identification by mass spectrophotometery using immunoprecipitation (IP) for enrichment. The BicC1 antibodies are currently being used for IP in human neuroblastoma cell lines. These IP will be subsequently run on western blotting gels to isolate and enrich specific bands to be sent for mass spectrophotometery analysis at the proteomics facilities at the IOP, king's college, London. 
 
Title RegSNP 
Description RegSNP is a new computer algorithm for the prediction of the effects of SNPs on transcription factor binding to DNA. The algorithm was released on line in the form of an easily usable web site. 
Type Of Material Improvements to research infrastructure 
Year Produced 2009 
Provided To Others? Yes  
Impact The use of RegSNP allowed us to predict the effects of SNPs identified within the regulatory regions under study. We are also aware of the use of this web site by other researchers overseas. 
 
Title Reporter gene transgenic lines 
Description The material consists of a number of trangenic lines that contain reporter gene constructs made with enhancers identified during the time frame of the current projecty 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2008 
Provided To Others? Yes  
Impact The material provided allow for the assessment of the tissue specific and inducible properties of enhancers identified by comparitive genomics in vivo. These novel models allow for the production of persuasive in vivo data on the properties of novel enhancers. The regulatory regions currently being modelled in these lines include enhancers and promoters for Galanin, TAC1, CGRP, NPY, BDNF and CNR1. 
 
Description Bioinformatic analysis, Virginia Tech 
Organisation Virginia Tech
Department Virginia Bioinformatics Institute
Country United States 
Sector Academic/University 
PI Contribution Harold Skip Garner, Director of Virignia Tech Bioinformatic Institute
Collaborator Contribution New skills set
Impact Functional analysis of genetic analysis defining important genetic variants associated with neuroscience and cancer.
Start Year 2011
 
Description Development of a novel Algorithm to define SNP effects on DNA binding sites 
Organisation University of Aberdeen
Department School of Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided intellectual input and have guided the project from the biological perspective.
Collaborator Contribution Dr Starkey has brought expertise in Computer software engineering.
Impact We have one paper in press (BMC genomics) and one to be submitted very soon in BMC computational biology that describes the development of a novel web site that allows researchers to predict the effects of non-coding polymorphisms on the binding of transcription factors
Start Year 2006
 
Description Regulation of hypothalamic galanin expression 
Organisation University of Aberdeen
Department Rowett Institute of Nutrition and Health Aberdeen
Country United Kingdom 
Sector Academic/University 
PI Contribution A collaboration to understand the systems that regulate the expression of Galanin in the PVN of the hypothalamus and the amygdala.
Collaborator Contribution Dr Perry Barrett brings expertise in hypothalamic function and in-situ hybridisation
Impact We have used comparitive genomics to identify a gene regulatory region that supports expression of the galanin gene in the hypothalamus, amygdala and in sensory neurones. We have also found that this enhancer is highly polymorphic ans that polymorphisms affect the ability of the enhancer to activate transcription of a marker gene in primar hypothalamic neurones. We are in the process of completeing our studies and a paper has been published in the highly respected journal Neuropsychopharmocology
Start Year 2007
 
Description Regulation of the CGRP gene 
Organisation University of Iowa
Department Department of Molecular Physiology and Biophysics
Country United States 
Sector Academic/University 
PI Contribution We made a transgenic line containing a reporter construct with the enhancer region previously identified and studied in the Russo lab. We also carried out immunohistochemical co-localisation between the activity of this enhancer and expression of the endogenous gene.
Collaborator Contribution Working with Prof Russo we have identified and generated transgenic models with a novel CGRP enhancer region. The activity of this enhancer replicates many of the expression characteristics of the endogenous cgrp gene and overlaps the expression of Calcetonin in the thyroid gland. This work is currently in preperation for submision to PLOS one.
Impact A manuscript is currently in preperation.
Start Year 2009
 
Description Shared Studentship 
Organisation National Center for Biotechnology Information (NCBI)
Country United States 
Sector Public 
PI Contribution We have a shared WT/NIH PhD student which is addressing DNA structure in areas of regulatory domains relevant for transcription
Collaborator Contribution We have a shared WT/NIH PhD student which is addressing DNA structure in areas of regulatory domains relevant for transcription
Impact We have a shared WT/NIH PhD student which is addressing DNA structure in areas of regulatory domains relevant for transcription
Start Year 2008
 
Description determining the mechanisms of BDNF enhancer polymorphisms on cognitive decline in the elderly. 
Organisation University of Manchester
Department School of Medicine Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We carried out the bioinformatic analysis, cloning and reproduction of the associated alleles using site directed mutatgenesis. We cloned promoter 4 of the BDNF gene and showed that this promoter drove marker gene expression in the limbic system including the amygdala and the hippocampus. Using luciferease assays in primary hippocampal cultures we were able to show that the associated polymorphism could significantly reduce the ability of the enhancer in which it was contained to activate promoter 4 in hippocampal neurones following depolarisation
Collaborator Contribution The University of Manchester provided association data for a SNP within a BDNF enahancer element that was associated with cognitive decline in the elderly. We have now show that this SNP affects the ability of the enhancer to activate BDNF promoter 4 in primary hippocampal neurones depolarised by KCl. Considering the important role played by BDNF in hippocampal plasticity the mechanistic linking of a cognitive disorder linked SNP and cell depolarisation in the hippocampus is highly significant.
Impact A paper describing this research has been invited for resubmission by Biological Psychiatry
Start Year 2008
 
Description GWA Studies, Gene Regulatory Variation and Disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Seminar/workshop

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Gene regulation, SNPs and disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Many interesting questions were asked and views shared

I hope to have stimulated young people in the audience to pursue science as a career
Year(s) Of Engagement Activity 2011,2012
 
Description Gene regulatory variation in health and disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Seminar/workshop

The talk stimulated a great deal of interest from the audience
Year(s) Of Engagement Activity 2011
 
Description Invited speaker to the Wellcome Trust, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was one of three invited speakers involved in presenting a Wellcome trust exhibition entitled "Gut Reactions" which aimed to inform the general public about research into the genetics of appetite. The occation generated a great deal of audience interest and many questions and was reported in the Observer.

The occation generated an article in the Observer and was also reported in a number of other newspapers.
Year(s) Of Engagement Activity 2011
 
Description Newspaper articles 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Articles published in the following news papers; PRESS & JOURNAL, THE SCOTTISH SUN, DAILY RECORD, THE SCOTSMAN, DAILY RECORD, METRO SCOTLAND, ABERDEEN EVENING EXPRESS, THE SCOTSMAN, THE HERALD (10.06.08)

I was subsequently invited to contribute to several radio programmes.
Year(s) Of Engagement Activity 2008
 
Description Radio programmes 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interviewed on BBC RADIO ABERDEEN - NORTH EAST NEWS, NORTHSOUND 2 - NEWS, BBC RADIO SCOTLAND - NEWSDRIVE (06.06.08)

Invites to take part in television programmes.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
 
Description RegSNP - Predicting Allele Specific Differences in Transcription Factor - DNA binding 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This website permits non-expert in biotechnology to identify the transcription factor bindingsites most affected by specific SNPs. The website also displays LD data, information on disease data and whether a particular SNP is GWAS associated or alters suceptibility to epigenetic modifiation.

Our web site has informed the research of many other researchers.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014
URL http://viis.abdn.ac.uk/regsnp/Home.aspx
 
Description School Visit-Glasgow 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I gave a presentation of the importance of gene regulation on disease and evolution.

I hope that I motivated the students to learn more about biology
Year(s) Of Engagement Activity 2008
 
Description Speaker at the Bradford Science festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Over 40 reporters from all of the main newspapers and a number of science based magasines were present. This resulted in the production of a number of blogs, newspaper articles in papers such as the Guardian and articles in a number of magazines.

The publication of the work sparked a great deal of media interest.
Year(s) Of Engagement Activity 2011
 
Description Television appearances 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Gave an interview about my work to BBC television.

Wider interest in my work.
Year(s) Of Engagement Activity 2008,2009