Identification of functional variants in the Alzheimer's disease candidate gene ABCA7. PhD thesis

284 Pages·2017·4.13 MB·English

by  Christine Middleton

Identification of Functional Variants in the
Alzheimer’s Disease Candidate Gene ABCA7

Naomi Susan Clement BMedSci (Hons)

Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy

March 2016
Mischief managed.

Mooney, Wormtail, Padfoot and Prongs
Harry Potter and the Prisoner of Azkaban
Abstract

Late onset Alzheimer’s disease (LOAD) is the commonest form of dementia, affecting
approximately 850,000 patients in the UK alone, predicted to exceed one million by 2025.
The cause of LOAD is complex, but several large Genome Wide Association Studies have
highlighted 21 genetic loci associated with this devastating disease and the ATP-Binding
Cassette Protein, family A, member 7 (ABCA7) is one of these genetic loci. However, the
exact reasons behind this association are still unknown, focusing work on identifying
functional, pathogenic mutations within this locus.
A total of 240 exonic variations within ABCA7 were therefore annotated in order to identify
ones potentially altering the functionality of ABCA7. A total of five variants were predicted
to be damaging by in silico annotation tools: rs3752233; rs59851484; rs3752237;
rs114782266 and a novel mutation at genomic position 19:1056958. These were genotyped in
the ARUK DNA Bank resource and three (rs59851484, rs3752239 and 19:1056958) showed
tentative association with LOAD. However, lack of power in this study prevented any
definitive associations from being formed. A further two variants were examined within
functional cell assays. rs881768 had been predicted to affect the splicing of the ABCA7
protein and appeared to do so within minigene cellular assays. However, this did not appear to
be the case when RNA from brain tissue harbouring this variation was examined. rs2020000
was examined through the dual luciferase assays, with the minor allele seeming to down
regulate the reporter protein by approximately 30% (p<0.02) in these in vitro assays.
Functional variations within the ABCA7 locus do play a role in LOAD risk and improvements
within functional databases and annotation programmes will assist in identifying these
causative mutations, in order to put a halt to LOAD, as well as other destructive complex
disorders.
Publications and Presentations
Publications
Clement, N., Medway, C., Carrasquillo, M. M., Guetta-Baranes, T. et al., Mapping
regulatory variants in the AD candidate gene ABCA7. Manuscript in preparation.
Clement, N., Braae, A., Turton, J., Lord, J. et al., November 2016. Investigating splicing
variants uncovered by next-generation sequencing the Alzheimer’s disease candidate
genes, CLU, PICALM, CR1, ABCA7, BIN1, the MS4A locus, CD2AP, EPHA1 and CD33.
J. Alzheimer’s Dis. Park.
Boden, K., Barber, I., Clement N., et al., October 2016. Methylation profiling of key genes
associated with sporadic early onset Alzheimer’s disease. Submitted to J. of Alzheimer’s
Dis.
Sassi, C., Guerreiro, R., …, Clement N.…., et al., October 2016. ABCA7 p.G215S as
potential protective factor for Alzheimer's disease. Neurobiol. Aging.
doi:10.1016/j.neurobiolaging.2016.04.004
Barber, I.S., Braae, A., Clement, N., et al., September 2016. Mutation analysis of sporadic
early-onset Alzheimer’s disease using the NeuroX array. Neurobiol. Aging.
Brookes K., Patel T., … Clement, N. …, et al., May 2016. Identifying Polymorphisms in
the Alzheimer's Related APP Gene Using the Minion Sequencer. Next Generat Sequenc &
Applic 3:125. doi:10.4172/2469-9853.1000125
Barber, I.S., García-Cárdenas, J.M., …, Clement, N.…., et al., March 2016. Screening
exons 16 and 17 of the amyloid precursor protein gene in sporadic early-onset Alzheimer’s
disease. Neurobiol. Aging 39, 220.e1-7. doi:10.1016/j.neurobiolaging.
2015.12.011
Presentations

“Examining Predicted Splice Variants in Late Onset AD Risk Loci.” Alzheimer’s Research
UK Conference 2015. Poster presentation, 9th-11th March 2015, London, UK.
“Mapping Regulatory Variants in the AD Candidate Gene ABCA7.” Alzheimer’s Research
UK Conference 2016. Poster presentation, 7th-9th March 2016, Manchester, UK.
“Mapping Regulatory Variants in the AD Candidate Gene ABCA7.” Alzheimer’s Association
International Conference 2016. Poster presentation, 24th-28th 2016, Toronto, Canada.
“Mapping Regulatory Variants in Late Onset AD Candidate Genes.” Alzheimer’s Research
UK Midlands Network Conference 2016. Oral Presentation, 13th September 2016,
Loughborough, UK.
“Mapping Regulatory Variants in the AD Candidate Gene ABCA7.” East Midlands Student
Research Conference 2016. Oral Presentation, 5th November 2016, Leicester, UK. Awarded
Best Oral Breakout Presentation.
“Mapping Regulatory Variants in the AD Candidate Gene ABCA7.” Genetics Society 2016
Autumn Meeting. Poster presentation, 10th-11th November 2016, London, UK.
Grants
Glomax 96 Microplate Luminometer, awarded to Naomi Clement and Professor Kevin
Morgan. Equipment Grant (£2000) received from ARUK East Midlands Network. January
2016.
Acknowledgements

First of all, I would like to acknowledge my supervisors Professors Kevin Morgan and
Mohammad Ilyas for their support and advice, especially Kevin for his endless advice,
positivity and (sometimes) humorous stories!
I am also indebted to the Jean Shanks Foundation for funding me throughout my PhD,
as well as ARUK for their support of the Human DNA and Brain Bank hosted in the
Human Genetics laboratory at the University of Nottingham. And of course, my
gratitude to all the patients and families who have agreed to donate their tissues as,
without them, none of this work would be possible.
My thanks also go to the students whose work has contributed in part to this thesis:
Sara Garin; Akili Mata and Ahmed Alahmad (MSc. Students) whose dissertation
works form part of Chapter 3. My appreciation also to the Molecular Diagnostics lab
for analysing all of the Sanger sequencing performed throughout this project.
Tamar Guetta-Baranes: I really couldn’t have done this without your constant advice,
teaching and, of course, the regular escape to your office. Thank you for everything.
To all the other members of the Human Genetics Group as well, thanks for providing
such a wonderful atmosphere in which to work: Anne Braae; Keeley Brookes; Tulsi
Patel and Sally Chappell all provided help, advice and, when needed, just a chat.
Particular thanks to Keeley and Anne for their comments on much of this thesis. I
would also like to thank James Turton for performing much of the ground work for
this thesis as well as introducing me to the wonderful world that is human genetics.
My unreserved thanks and love to my family whom, again, I couldn’t have done this
without. Thanks to Mum and Dad for providing me so many opportunities throughout
my life and allowing me to take this one. My total gratitude also goes to Emma, Nana
and Helen for always being there for me, no matter what. I love you all
unconditionally. Mentions as well, to the friends who sometimes knew not to ask
questions and just provide more wine: Abigail; Jack and Adam.
And finally to Rob, for being my rock. I don’t know what I did to deserve you but I
am so glad you pushed me to do this, stuck with me throughout the roller coaster this
thesis was and believed in me, even when I didn’t myself. I hope one day I can even
partially return the favour.
Table of Contents
List of Abbreviations ................................................................................................... 1
1 Introduction .......................................................................................................... 6
1.1 Alzheimer’s Disease..................................................................................... 6
1.2 Genome Wide Association Studies ............................................................ 30
1.3 ABCA7 ...................................................................................................... 36
1.4 The ABC Family and Disease .................................................................... 53
1.5 ABCA7 and Disease .................................................................................. 56
1.6 ABCA7 and Alzheimer’s Disease Pathways .............................................. 59
1.7 Study Aims................................................................................................. 62
2 Examining ABCA7 Exonic Variants ................................................................... 63
2.1 Introduction & Background........................................................................ 63
2.2 Methods ..................................................................................................... 69
2.3 Results ........................................................................................................ 75
2.4 Discussion .................................................................................................. 84
2.5 Conclusions ................................................................................................ 92
3 Case-Control Genotyping of Putative Damaging ABCA7 Variants .................... 93
3.1 Introduction ................................................................................................ 93
3.2 Materials & Methods .................................................................................. 97
3.3 Results ...................................................................................................... 109
3.4 Discussion ................................................................................................ 118
3.5 Conclusions .............................................................................................. 125
4 Minigene Splicing Assays ................................................................................ 126
4.1 Introduction .............................................................................................. 126
4.2 Materials & Methods - Minigene Assay ................................................... 139
4.3 Materials & Methods - RNA Extraction from Brain ................................ 153
4.4 Results ...................................................................................................... 155
4.5 Discussion ................................................................................................ 162
4.6 Conclusions .............................................................................................. 167
5 Nonsense Mediated Decay ............................................................................... 168
5.1 Materials & Methods ................................................................................ 172
5.2 Results ...................................................................................................... 175
5.3 Discussion ................................................................................................ 181
5.4 Conclusions .............................................................................................. 182
6 Dual Luciferase Assays .................................................................................... 183
6.1 Introduction & Background...................................................................... 183
6.2 Materials & Methods ................................................................................ 192
6.3 Results ...................................................................................................... 207
6.4 Discussion ................................................................................................ 214
6.5 Conclusions .............................................................................................. 222
7 General Discussion .......................................................................................... 223
7.1 AD Genetics Update ................................................................................ 228
7.2 Future of AD Genetics ............................................................................. 230
7.3 Conclusions .............................................................................................. 232
8 References ........................................................................................................ 233
Appendix A .............................................................................................................. 265
Appendix B - URLs Used ........................................................................................ 271
Table of Figures
Figure 1.1 - Comparison between a healthy brain and a brain with Alzheimer's
disease.......................................................................................................................... 6
Figure 1.2 - Comparison between a healthy neuronal cell and a neuronal cell from an
individual with AD pathology. ..................................................................................... 8
Figure 1.3 - Amyloid Precursor Protein proteolysis. .................................................. 10
Figure 1.4 - Amyloid Cascade Hypothesis. ................................................................ 12
Figure 1.5 - Disease pathways implicated in AD based on genetic studies. ............... 35
Figure 1.6 - Genetic summary of the ABCA7 gene. ................................................... 38
Figure 1.7 - Schematic of an ABCA full-size transporter. ......................................... 39
Figure 1.8 - Tissue specific expression for ABCA7 mRNA. ....................................... 43
Figure 2.1 - Bioinformatics pipeline used to analyse NGS data. ................................ 68
Figure 2.2 - Breakdown of all variants catalogued from EVS and NGS data. ............ 75
Figure 2.3 - Output from QUANTO upon performing power calculations. ............... 77
Figure 2.4 - Results from the LD calculations performed between the variants of
interest and the ABCA7 GWAS tag SNP. ................................................................... 80
Figure 2.5 - 2D representation of ABCA7's structure within the cell membrane
including the location of the variants of interest. ....................................................... 81
Figure 2.6 - Prediction of the affect the variant at position 19:1056958 will have on
ABCA7's transmembrane domains. ........................................................................... 82
Figure 2.7 - 2D representation of ABCA1's structure within the cell membrane. ...... 91
Figure 3.1 - Methodology of the KASP genotyping assay. ........................................ 96
Figure 3.2 - Examples of the file formats used to statistically analyse the genotyping
results in PLINK. ..................................................................................................... 107
Figure 3.3 - Example of a dual colour scatter plot allowing genotyping of samples for
variant rs3752239. ................................................................................................... 111
Figure 3.4 - Example of a dual colour scatter plot allowing genotyping of samples for
variant at position 19:1056958. ................................................................................ 112
Figure 3.5 - Exmple of an agarose gel showing amplification of samples genotyped
for rs3752233. .......................................................................................................... 113
Figure 4.1 - cis-sequences vital to successful splicing. ............................................ 128
Figure 4.2 - Three main steps involved in eukaryotic mRNA splicing..................... 129
Figure 4.3 - Consequences of alternative splicing. ................................................... 131
Figure 4.4 - Methodology behind the minigene assay. ............................................. 134
Figure 4.5 - In silico predictions of the splicing variant rs881768 in exon 32 of the
ABCA7 gene. ............................................................................................................ 138
Figure 4.6 - Agarose gel showing samples amplified to ascertain their genotype for
varinat rs881768. ..................................................................................................... 156
Figure 4.7 - Agarose gel showing plasmid DNA clones created for varinat rs881768.
................................................................................................................................. 156
Figure 4.8 - cDNA synthesised from four seperate transfections into COS-7 cells. . 158
Figure 4.9 - cDNA synthesised from three seperate transfections into BE(2)-C cells.
................................................................................................................................. 159
Figure 4.11 - Alignment of band only present in samples containing the minor allele.
................................................................................................................................. 160
Figure 4.10 - Alignment of the band present in all samples. .................................... 160
Figure 4.12 - Agarose gel and alignment of cDNA amplified from brain tissue
samples carrying both genotypes of rs881768. ........................................................ 161
Figure 5.1 - cis-sequences required to initiate nonsense mediated decay. ................ 169
Figure 5.2 - 12 well plate created to examine the affect of nonsesne mediated decay on
the ABCA7 isoform. ................................................................................................ 173
Figure 5.3 - Example of an agarose gel depicting the affect of nonsense mediated
decay inhibition on the ABCA7 isoform produced. ................................................. 176
Figure 5.4 - Electropherogram showing the affect of nonsense mediated decay
inhibition on the ABCA7 isoform. ........................................................................... 177
Figure 5.5 - Electrohpherogram showing the positive control used to unsure nonsense
mediated decay had been inhibited in these experiments. ........................................ 179
Figure 5.6 - Sequencing results of this positive control to show nonsense mediated
decay had been inhibited. ......................................................................................... 180
Figure 6.1 - Representation of the genomic position of rs2020000 showing its’
relationship with other ABCA7 variants. .................................................................. 189
Figure 6.2 - Genomic region of rs2020000 showing the important regulatory elements
present...................................................................................................................... 190
Figure 6.3 - Primer sequences added in order to utilise the Gateway cloning system.
................................................................................................................................. 194
Figure 6.4 - Representation of the Gateway cloning system used. ........................... 197
Figure 6.5 - Circle maps of the pGL3 vectors. ......................................................... 201
Figure 6.6 - Agarose gel showing the samples amplified to determine their genotype
for variant rs2020000. .............................................................................................. 208
Figure 6.7 - LD plot between rs2020000 and rs10419707. ...................................... 209
Figure 6.8 - Agarose gel showing the vectors used in the dual-luciferase assays
performed................................................................................................................. 210
Figure 6.9 - Median values for the pGL3-Basic vector. ........................................... 211
Figure 6.10 - - Median values for the pGL3-Promoter vector. ................................. 212
Figure 6.11 - - Median values for the pGL3-Enhancer vector. ................................. 212
Figure 6.12- Single tissue expression data for rs2020000 from the GTEx database.215
Figure 6.13 - Transcription factor binding sites around the region of rs2020000. .... 221