The association of specific polymorphisms in the serotonergic system with aggressive, impulsive ...

189 Pages·2016·3.51 MB·English

The association of specific polymorphisms in the
serotonergic system with aggressive, impulsive and
suicidal behaviour

Susan Louw

Dissertation presented in order to qualify for the degree Magister Scientiae in Behavioural
Genetics in the Faculty of Natural and Agricultural Sciences, Department of Genetics, at
the University of the Free State

June 2016
Supervisor: Prof. J. J. Spies
Co-supervisors: Ms. L. J. Heathfield, Ms. S. R. Schneider

The financial assistance of the National Research Foundation (NRF) towards this research is hereby
acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not
necessarily to be attributed to the NRF.
Declaration

I, Susan Louw, declare that the dissertation hereby submitted for the Master’s degree
qualification, M.Sc in Behavioural Genetics at the University of the Free State is my own
independent effort and had not previously been submitted for a qualification at another
University/Faculty.

I, Susan Louw, furthermore waive copyright of the dissertation in favour of the University of
the Free State. All royalties with regards to intellectual property that was developed during
the course and/or in connection with the study at the University of the Free State will
accrue to the University.

_____________________ ___________________
S. Louw Date

1
Acknowledgements

I would like to take this opportunity to thank everyone that contributed in some way
to this study and the experience I gained from it, be it in a small, large, positive or negative
way.

Special thanks go to Laura Heathfield for her invaluable comments, support,
encouragement, and time. Her input was vital to the completion of this study and I really
appreciate it. I would like to extend my gratitude to Prof. Schall from the Department of
Actuarial Sciences, University of the Free State, for his invaluable contribution to the
statistics of this project. Also to Prof. Spies, who agreed to act as my study leader, for his
input.

I would like to thank the National Research Foundation for the financial support I
received, and the Department of Genetics at the University of the Free State, for their
support and infrastructure. Without the participants, I could not have done this study, so to
each of the anonymous participants, thank you.

Then to all those non-academic people who made coffee; listened to my grievances
and troubles; gave advice; sometimes even read a paragraph; and were just there: thank
you so much. In little ways you all made this easier and I appreciate it.

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Table of Contents

Declaration .............................................................................................................................................. 1
Acknowledgements ................................................................................................................................. 2
Table of Contents .................................................................................................................................... 3
List of Tables ........................................................................................................................................... 6
List of Figures .......................................................................................................................................... 8
List of abbreviations ................................................................................................................................ 9
Abstract ................................................................................................................................................. 13
Opsomming ........................................................................................................................................... 15
Chapter 1 Motivation ............................................................................................................................ 17
1.1 Introduction .................................................................................................................................... 17
1.2 Background ..................................................................................................................................... 18
1.3 Research statement ........................................................................................................................ 24
1.4 Aim of this study ............................................................................................................................. 24
1.5 Objectives of this study ................................................................................................................... 25
1.5.1 Theoretical objectives .............................................................................................................. 25
1.5.2 Empirical objectives ................................................................................................................. 26
1.6 Research outline of this study ......................................................................................................... 26
Chapter 2 Literature Review ................................................................................................................. 28
2.1 Background ..................................................................................................................................... 28
2.2 Suicide ............................................................................................................................................. 29
2.2.1 Burden of suicide ..................................................................................................................... 29
2.2.2 Definition of suicide ................................................................................................................. 33
2.3 Suicidal behaviour from a behavioural viewpoint .......................................................................... 34
2.3.1 Factors influencing suicidal behaviour ..................................................................................... 34
2.3.2 Clinical model of suicidal behaviour ........................................................................................ 36
2.4 Suicide from a genetics viewpoint .................................................................................................. 38
2.4.1 Family genetic studies .............................................................................................................. 39
2.4.2 Twin studies ............................................................................................................................. 40
2.4.3 Adoption studies ...................................................................................................................... 41
2.5 Endophenotypes of suicidal behaviour ........................................................................................... 42
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2.5.1 Impulsivity ................................................................................................................................ 44
2.5.2 Aggression ................................................................................................................................ 46
2.5.3 Impulsive aggression and suicidal behaviour .......................................................................... 48
2.6 Neurotransmitter pathways and behaviour ................................................................................... 50
2.7 Serotonin and suicidal behaviour ................................................................................................... 53
2.7.1 Serotonergic pathway .............................................................................................................. 53
2.7.2 Serotonin and behaviour ......................................................................................................... 55
2.7.3 Serotonergic genes .................................................................................................................. 59
2.8 Remarks .......................................................................................................................................... 64
Chapter 3 Materials and Methods ........................................................................................................ 66
3.1 Research design and methodology ................................................................................................. 66
3.2 Cohort ............................................................................................................................................. 66
3.3 Ethical considerations ..................................................................................................................... 68
3.4 Quantitative analysis ....................................................................................................................... 69
3.4.1 Questionnaires ......................................................................................................................... 69
3.4.2 Statistical analysis .................................................................................................................... 73
3.5 Molecular genetic analysis .............................................................................................................. 74
3.5.1 DNA extraction and quantification .......................................................................................... 75
3.5.2 Gene and SNP selection ........................................................................................................... 75
3.5.2 Polymerase Chain Reaction (PCR) ........................................................................................... 76
3.5.2 Genotyping ............................................................................................................................... 77
3.5.4 Statistical analysis ........................................................................................................................ 80
Chapter 4 Results .................................................................................................................................. 82
4.1 Quantitative analysis ....................................................................................................................... 82
4.1.1 Cronbach’s alpha ...................................................................................................................... 82
4.1.2 Difference between impulsivity and aggression for the suicide attempters and control group
.......................................................................................................................................................... 83
4.1.3 Correlations between impulsivity and aggression scales ........................................................ 85
4.1.4 Reactive aggression in the suicide group ................................................................................. 88
4.2 Molecular analysis........................................................................................................................... 89
4.2.1 Genotyping ............................................................................................................................... 89
4.2.2 Statistical analysis .................................................................................................................... 92
4.3 Trends observed between impulsivity and aggression measures and respective genotypes
..............................................................................................................................................................97
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Chapter 5 Discussion ........................................................................................................................... 100
5.1 Cohort ........................................................................................................................................... 101
5.1.1 Gender and suicidal behaviour .............................................................................................. 103
5.1.2 Race and suicidal behaviour .................................................................................................. 104
5.1.3 Age and suicidal behaviour .................................................................................................... 106
5.2 Quantitative analysis ..................................................................................................................... 107
5.2.1 Impulsivity, aggression and suicidal behaviour ..................................................................... 107
5.2.2 Impulsive aggression and suicidal behaviour: clarification of terms ..................................... 111
5.3 Molecular analysis......................................................................................................................... 114
5.3.1 Genotype and allele frequencies ........................................................................................... 115
5.3.2 The association between impulsivity and aggression in suicidal behaviour with serotonergic
genetic variants ............................................................................................................................... 122
Chapter 6 Conclusion .......................................................................................................................... 128
References .......................................................................................................................................... 134
Appendix A ............................................................................................................................................ 13
Appendix B .......................................................................................................................................... 165
Appendix C .......................................................................................................................................... 167
Appendix D .......................................................................................................................................... 169
Appendix E ............................................................................................... Error! Bookmark not defined.
Appendix F .......................................................................................................................................... 179
Appendix G .......................................................................................................................................... 180
Appendix H .......................................................................................................................................... 182
Appendix I ........................................................................................................................................... 183
Appendix J ........................................................................................................................................... 184
Appendix K .......................................................................................................................................... 185
Appendix L ........................................................................................................................................... 186

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List of Tables

Table 2.1: Genetic variants implicated in suicidal behaviour..................................................50
Table 3.1: Factor structure and scoring of the Barratt Impulsiveness Scale (BIS-11)..............71
Table 3.2: Genes and SNPs selected for this study.................................................................76
Table 3.3: Forward and reverse primers with their respective properties.............................77
Table 3.4: Digestion sites of the restriction endonucleases for each SNP..............................79
Table 4.1: Summary of mean scores, standard deviation, median values, and Cronbach’s
alpha for BIS-11, BPAQ, and RPQ, and respective statistical analysis.....................................84
Table 4.2: Observed partial Pearson correlations between BIS-11 impulsivity and RPQ and
BPAQ aggression measures in the suicide and control group.................................................86
Table 4.3: Allele and genotype frequencies presented for each SNP and respective statistical
analysis....................................................................................................................................93
Table 4.4: Mean and standard deviation for the BIS-11, RPQ, and BPAQ, stratified for each
genotype, with statistical analysis...........................................................................................97
Table 4.5: Trends observed for the questionnaire mean response stratified by genotype.p.98
Table A.1: Typical cycling conditions for the PCR of each gene region amplified according to
optimised in-house protocols...............................................................................................179
Table A.2: Genotypes for the suicide attempters and control group for HTR1A, HTR1B,
HTR2A, and SLC6A4...............................................................................................................184
Table A.3: Hardy-Weinberg equilibrium for each genetic variant........................................185
Table A.4: Suicide attempters – mean response and standard deviation for each scale of the
questionnaire, stratified by genotype, with statistical analysis for genotype comparisons
between scores.....................................................................................................................186
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Table A.5: Control group – mean response and standard deviation for each scale of the
questionnaire, stratified by genotype, with statistical analysis for genotype comparisons
between scores.....................................................................................................................187
Table A.6: Total cohort – mean response and standard deviation for each scale of the
questionnaire, stratified by genotype, with statistical analysis for genotype comparisons
between scores.....................................................................................................................188

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List of Figures

Figure 2.1: Suicide by age as presented by the National Injury Mortality Surveillance
System.....................................................................................................................................31
Figure 2.2: A clinical model of suicidal behaviour...................................................................37
Figure 2.3: Endophenotype markers for suicidal behaviour...................................................53
Figure 2.4: A schematic view of serotonergic neurotransmission..........................................55
Figure 2.5: Chosen variants in the serotonergic pathway and resultant maladaptive
behaviour................................................................................................................................64
Figure 3.1: A box plot of the observed range and mean ages of the suicide attempters and
the control group....................................................................................................................67
Figure 4.1: ANCOVA plots graphically illustrating the comparison of the relationship between
the scales for suicide and control group.................................................................................88
Figure 4.2: Representation of the verification of the correct PCR products for HTR1A through
agarose gel electrophoresis....................................................................................................90
Figure 4.3: HTR2A DNA fragments that underwent MspI digestion.......................................91
Figure 4.4: Mean and standard deviation for the BIS-11, RPQ, and BPAQ stratified for each
genotype and statistical analysis.............................................................................................94
Figure 4.5: Trends observed for the questionnaire mean response stratified by
genotype.................................................................................................................................95
Figure A.1: Molecular weight marker....................................................................................182
Figure A.2: Agarose gel of the HTR1A DNA fragments that underwent BseGI digestion......183
Figure A.3: Agarose gel of the HTR1B DNA fragments that underwent HincII digestion......183
Figure A.4: Agarose gel of the SLC6A4 HTTLPR.....................................................................183
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List of abbreviations

5-HIAA 5-hydroxyindoleacetic acid
5-HT 5-hydroxytryptamine/serotonin
5-HTT serotonin transporters
5-HT1A serotonin receptor 1A
5-HT1B serotonin receptor 1B
5-HT2A serotonin receptor 2A
α alpha
β beta
χ² chi square
°C degrees Celsius
= equals
< less than
% percent
μg microgram
μl microliter
μM micro molar
A adenine
ADHD attention deficit hyperactivity disorder
APA American Psychological Association
ANCOVA analysis of covariance
BIS-11 Barrat Impulsiveness Scale version 11
BLAST Basic Local Alignment Search Tool
bp base pair
BPAQ Buss Perry Aggression Questionnaire
BDHI Buss Durkee Hostility Inventory
BSA bovine serum albumin
C cytosine
CDC Centre for Disease Control
CI confidence interval
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