1. Targeted Next Generation Sequencing (tNGS) in Anxiety Disorders
Mr NW McGregor1, Dr J Dimatelis2, Dr SMJ Hemmings1, Dr CJ Kinnear3,6, Prof DJ Stein4,5, Prof V RusselL2, Prof C Lochner5
1Department of Psychiatry, University of Stellenbosch; 2Department of Human Biology, University of Cape Town; 3Division of Molecular Biology and Human Genetics, University of Stellenbosch; 4Department of Psychiatry and Mental Health, University of Cape Town, 5MRC Unit on Anxiety and Stress Disorders1, University of Stellenbosch, 6MRC Centre for Molecular and Cellular Biology
INTRODUCTION
Large scale studies performed in the 1990s and early 2000s have shown that anxiety disorders are more common than originally thought and are debilitating and costly in both the developed and developing world. South Africa has a high prevalence rate for any anxiety disorder at 15.8 % (approximately 6.4 million people), with a lifetime prevalence rate of 30.1 % (approximately 12.4 million people).
Anxiety aetiology remains poorly understood. Clear evidence for a genetic component has been proposed; however, there also is an increasing focus on the genetic influences and environmental factors, and the interaction between them that may mediate the pathogenesis of anxiety disorders.
Anxiety disorders are considered of the world’s most debilitating conditions
South Africa has of the highest prevalence rates globally, with a life-time prevalence of 30.1 %
The aetiology of anxiety disorders remains incompletely understood
Clear evidence for a genetic component exists but increasing weight is being put on genetic and environmental influences and their interaction in disorder pathogenesis

2. THIS STUDY
Genes previously identified to be aberrantly expressed (relative to controls) in a maternally and/ or restraint-stressed Sprague Dawley rat model were subjected to tNGS (whole-gene amplification and deep sequencing) to assess for novel and previously identified polymorphisms in a South African Caucasian population.
Identified polymorphisms were screened in an extended anxiety disorders cohort (obsessive-compulsive disorder (OCD), panic disorder (PD) and social anxiety disorder (SAD) in a case-control association fashion.
AIMS
To identify novel candidate genes that may play a role in anxiety disorders in a South African population
To test for association in an extended anxiety disorders cohort for which a history of early-life trauma is known [childhood trauma questionnaire (CTQ)]
In this study genes previously identified to be aberrantly expressed in rats exhibiting anxiety-like behaviours after subjection to environmental interventions were subjected to targeted NGS
Polymorphisms identified were screened in an extended cohort of OCD, PAD and SAD diagnosed patients and controls and assessed for significance in a case-control association fashion (considering early-life trauma as an environmental contributor)

3. MATERIALS AND METHODS
Previously rats were subjected to maternal separation and restraint stress
Rats were behaviourally assessed and found to exhibit anxiety-like behaviours relative to control specimens
Rats were sacrificed and the striatal regions excised
RNA was extracted and synaptic plasticity assessed using PCR array technology
Genes identified to be aberrantly expressed were considered candidate susceptibility genes for anxiety disorders
Human homologues were amplified and subjected to tNGS using Illuminia’s MiSeq platform
Prioritised polymorphisms were screened for in an extended population using KASP genotyping technology in case-control fashion considering CTQ scale data as a covariate

4. Fold change in MAF (+/-)
RESULTS & DISCUSSION
6.4 G data: > 75% Q-Score > 30 (error rate of 1 in 1000)
103 SNPS and 4 INDELs (12 pts (OCD, PD and SAD), 19 cntrls)
38 unique to either only patients or only controls
83.3% of these were novel
CTQ data (pnts & cntrls) and genotyping in extended cohort
CTQ Patients
CTQ Controls
P <
Cohort
SNPS (%)
Indels (%)
Database
Intron
Exon
UTR
Patient
12 (32)
2 (50) 4
 12 1
Controls
26 (68) 9 10
dbSNP
SAMPLE
Type
Mut n
MAF
MAF dbSNP or Genomes
Fold change in MAF (+/-)
Gene
P - value rs
Patient
SNP
G->GA
185
0.04
0.12
- 3X
NTF4
0.575
novel
T->TG
182
0.02
N/A
GRM2
0.744 rs
C->CT
184
0.0006
+ 333X
0.862 rs
178
0.05
0.011
+ 4.5X
0.440 rs
Control
181
0.06
0.019
+ 3.15X
0.723 rs
186
0.01
+ 2X
0.464 rs
T->TC
0.14
- 3.5X
EGR2
0.323
6.4 Gigabases of data was generated of which >75% had excellent Q-scores
In the five target genes we found 103 SNPs and 4 indels considering 12 patients and 19 control individuals
Of these 38 were unique to either only patient or control subjects
Of these 83% were novel
Furthermore: more variation was found to be present in the control individuals (68%) than patients
And of this variation the majority was found to be exonic, as well in the UTR which could have transcriptional implications
Considering CTQ: A highly significant P value was obtained indicating significantly higher CTQ scale (and therefore early-life trauma) in patients compared to controls
Of the 38 polymorphisms screened in the extended cohort, 7 were found to be previously characterised
Although no significant P-values were obtained, substantially different MAFs obtained considering 1000 Genomes, dbSNP and Ensembl database entries

5. CONCLUDING REMARKS Of the genetic variants assessed, 83% were novel
unique nature of the South African population
warranting application of NGS in the identification of novel pathways, genes or polymorphisms that may be associated with complex disorders.
Results here indicate that common variants represent a rather small effect size and that the genetic architecture of anxiety-related disorders are most likely the result of a combination of common and rare variation.
Substantially different MAFs were observed compared to previously described data (1000 Genomes, Ensembl and dbSNP).
Small sample sizes utilized in this study may contribute to the non-significant P-values observed despite large MAF discrepancies. In anxiety disorders, the genetic components may be spread across several loci and therefore large sample sizes are necessary to assess a (most likely) small contribution of multiple genes and/ or polymorphisms to disorder pathogenesis.
A highly significant P-value (P < ) was found to be associated with the presence of childhood trauma in anxiety disorder patients providing merit to an environmental contribution to the pathogenesis of these disorders.
The results represented here warrant, at the very least, further consideration for NGS application in the anxiety disorders.
Novelty here attests to the unique nature of the South African population
NGS could be an important platform for pathway, gene and/ or mutation discovery in complex disorders such as anxiety disorders
Results suggest that common variants could present a rather small effect and that the genetic architecture of anxiety disorders are most likely a result of a combination of rare and common variation
Although no significant P-values: could just be attributed to small samples sizes
Genetic components of anxiety disorders could be spread across several loci so large samples sizes are necessary to identify association
Highly significant P-value associating early-developmental trauma with patients support literature findings suggesting it as a susceptibility risk in anxiety pathogenicity
Results warrant, at the very least, further consideration of NGS application in anxiety disorders research

6. ACKNOWLEDGMENTS National Research Foundation (NRF) – Funding
MRC Unit on Anxiety and Stress Disorders
Molecules and Genes in Inherited Conditions (MAGIC) Lab, 4036, Tygerberg Medical Campus